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Rous-Whipple Award Lecture |
From the Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
"Chance favors the prepared mind"
Louis Pasteur
Solid tumors are composed of two distinct but interdependent compartments: the malignant cells themselves (parenchyma) and the supporting connective tissue (stroma) that they induce and in which they are dispersed.1,2 The quantity of stroma is highly variable from tumor to tumor but all solid tumors, regardless of type or cellular origin, require stroma for nutrition and waste disposal if they are to grow beyond minimal size.3
The discrete separation of parenchymal cells from stroma is not unique to tumors. Normal tissues are also comprised of avascular parenchyma that abuts on vascularized connective tissue stroma. However, tumor stroma differs strikingly from normal connective tissue. Blood vessels offer one example. Tumor vessels differ from their normal counterparts with respect to organization, structure and function.2,4-7 Unlike the normal vasculature, tumor vessels are not arranged in a hierarchical pattern but are instead irregularly spaced and structurally heterogeneous. They are also hyperpermeable to plasma and plasma proteins, may lack pericytes, and are lined by actively dividing endothelial cells. Other elements of tumor stroma are also abnormal: increased amounts of plasma protein-rich interstitial fluid; structural proteins not normally found in mature stroma such as fibrin, tenascin, and fetal forms of fibronectin; abnormal proteoglycans; variable numbers of inflammatory cells, etc.
Given these extensive differences in composition, it seemed likely to us that tumor and normal stroma were formed by different steps and mechanisms. Although normal stroma generation is not completely understood, it is known to require the cooperative activities of many different cytokines and inhibitors, each expressed in appropriate amounts and temporal sequence.8-10 Among these are vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A), other members of the VPF/VEGF family such as placenta growth factor (PlGF), and other cytokines such as TGF-ß, PDGF, ephrins, FGFs, angiopoetins-1 and -2, and their respective receptors and inhibitors. By contrast, the imperfect nature of tumor stroma suggested to us that it was generated by a simpler process, one that deviated from normal developmental stromagenesis by having fewer steps and involving fewer cytokines.
The work reported here summarizes our progress over the past 25 years toward elucidating the steps and mechanisms of tumor angiogenesis and stromagenesis. A major conclusion is that a single cytokine, the 164/5 amino acid splicing variant of VEGF-A, is capable of inducing the formation of tumor-like blood vessels and connective tissue stroma. Further, VEGF-A164/5 is expressed not only by malignant tumors but also in healing wounds, in cellular immunity, and in chronic inflammatory diseases such as psoriasis and rheumatoid arthritis.4,5 Therefore, VEGF-A164/5 provides a unifying principle that contributes importantly to many, if not all, examples of pathological angiogenesis and tissue repair.
Discovery of Vascular Permeability Factor/Vascular Endothelial Growth Factor (VPF, VPF/VEGF, VEGF-A)
VEGF-A was discovered as the result of microscopic studies of transplantable tumors at early stages after their implantation into syngeneic animals.11,12
The critical observation was that tumors began as clumps of neoplastic cells dispersed in a fibrin gel stroma (Figure 1, a–d)
. Fibrin results from the clotting of plasma fibrinogen. Fibrinogen, like other plasma proteins, is normally retained within blood vessels; therefore, the presence of extravascular fibrin deposits strongly implied that tumor blood vessels were hyperpermeable to fibrinogen and presumably therefore to other plasma proteins as well. To test this hypothesis, we injected various macromolecular tracers intravenously into tumor-bearing animals and found extensive tracer extravasation from tumor as compared with normal blood vessels (Figure 1, e and f)
. Several earlier papers had also commented on the increased permeability of tumor blood vessels (reviewed in5
).
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. We called this activity vascular permeability factor (VPF). VPF was found to be non-dialyzable, largely inactivated by heat, and both cold and puromycin profoundly depressed its appearance in culture medium. Also, the vascular permeabilizing activity of tumor culture supernatants was not inhibited by antihistamines nor were these supernatants able to degranulate or release histamine from basophilic leukocytes or mast cells.12 We concluded from these studies that the VPF activity present in tumor culture supernatants and ascites fluids was a secreted protein and one that increased vascular permeability by acting directly on blood vessel endothelium.
Using the Miles assay to screen column fractions for their ability to induce vascular permeability, Donald Senger then purified VPF to homogeneity.13,14 Sometime later, our colleagues at the Monsanto Company15,16 made use of Senger’s N-terminal amino acid sequence data to clone VPF and show that it induced angiogenesis; independently, Napoleon Ferrara and his colleagues17 at Genentech cloned VPF and reported that it was mitogenic for cultured endothelial cells. As a result of these studies, VPF has come to be known more widely as vascular endothelial growth factor (VEGF) and, more recently, as VEGF-A.
VEGF-A and Its Activities
Over the past two decades much has been learned about VEGF-A.4,5,18 It is the founding member of a family of structurally related proteins with varying degrees of vasculogenic, angiogenic, and lymphangiogenic activity.4,5,18,19,20 The VEGF-A gene is alternatively spliced to yield major isoforms of 189, 165, and 121 amino acids; the murine homologues are in each case one amino acid shorter. Of these, the 164/5 isoform is generally expressed most abundantly. However, all three isoforms are essential for normal development and inactivation of even a single copy of the VEGF-A gene results in embryonic death from failure of vascular development.18,21,22 VEGF-A164/5 is expressed by the vast majority of malignant animal and human tumors,4,5 and interference with its activity, as by blocking antibodies or soluble receptors, inhibits tumor angiogenesis and tumor growth and may cause pre-existing tumors to regress.18
VEGF-A164/5 is a multifunctional cytokine with a vascular permeabilizing activity some 50,000 times more potent than histamine. It also stimulates endothelial cell migration, is mitogenic for endothelial cells and is active in standard angiogenesis assays. Important for these activities is its ability to reprogram endothelial cell gene expression, leading to the up-regulation of many proteins including various proteases, tissue factor, a glucose transporter, etc. VEGF-A164/5 is also an endothelial cell survival factor, able to avert both endothelial cell apoptosis and senescence.23,24 As far as is known, all of the activities of VEGF-A164/5 are mediated through two receptor tyrosine kinases (VEGFR-1, flt-1) and VEGFR-2 (flk-1, KDR), and through a more recently described, non-tyrosine kinase receptor, neuropilin.19,25
Expression of VEGF-A164 in Vivo
To investigate its capacity to induce tumor-like blood vessels and stroma, we sought to express VEGF-A164 in normal mouse tissues at a constant rate for a sufficient period of time to induce angiogenesis and new stroma. To that end we made use of a non-replicating adenoviral expression vector.26 Such vectors are readily engineered and offer a number of advantages for investigating the potential functions of angiogenic cytokines. 1) They can be injected in any accessible tissue of adult or embryonic mice where they direct infected host cells to express gene inserts. 2) Protein expression levels are readily adjusted by varying the dose of injected virus. 3) Control vectors engineered to express non-inflammatory proteins give insignificant backgrounds. 4) Combinations of cytokines can be studied, together or in sequence, by injecting vectors engineered to express different genes. 5) Adenoviral vectors are not integrated into the host cell genome and so the genes they introduce are not expressed indefinitely; this was actually an advantage for our purposes because it permitted us to study the effects of cytokine withdrawal on newly formed blood vessels and stroma.
The Vascular Response to VEGF-A164 Expression
Within hours of injection into any of several different tissues of nude mice or rats, infected host cells began to express VEGF-A164 mRNA and continued to do so at steady levels for 10 to 14 days, generating a strong angiogenic response (Figure 2)
;26
thereafter, VEGF-A164 expression declined gradually and, by 4 to 6 weeks was no longer detectable by in situ hybridization.
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The initial response (1 to 3 days) was similar in all tissues studied and consisted of vascular hyperpermeability, fibrin deposition, and edema.26 Generation of an extravascular fibrin gel requires not only leaky blood vessels but also activation of the clotting system. Clotting is triggered in most tissues within minutes of plasma leakage by tissue factor, a procoagulant expressed on tumor cells and many normal tissue cells.27,28 Tissue factor reacts with clotting factor VIIa and subsequently with other extravasated plasma-clotting proteins, culminating in thrombin generation, clotting of fibrinogen to fibrin, and subsequent cross-linking of fibrin by activated factor XIII. Once deposited, cross-linked fibrin behaves as a gel that causes edema by trapping extravasated plasma and provides a proangiogenic provisional stroma similar to that observed in transplanted tumors and healing wounds.1,11,12,29
The fibrin deposited in tissues by tumors or by Ad-VEGF-A164 represents a net balance, at any moment of time, between fibrinogen influx, clotting and fibrinolysis. Fibrinogen influx results from leakage of plasma from permeabilized vessels and clotting from activation of the tissue factor pathway. However, the plasma protein plasminogen also extravasates from leaky blood vessels where plasminogen activators cleave it to generate plasmin, a potent fibrinolytic protease. VEGF-A164 selectively up-regulates plasminogen activator activity in endothelial cells, and tumors commonly express this enzyme so that deposited fibrin is continually being remodeled.30
VVOs: The Principle Pathway for Macromolecule Extravasation from Hyperpermeable Blood Vessels
To investigate the anatomical pathway(s) by which macromolecules extravasated from Ad-VEGF-A164 permeabilized vessels, we injected anionic ferritin intravenously as a tracer. Ferritin is an iron-rich plasma protein that can be visualized directly by electron microscopy. In agreement with earlier studies of tumor vessels31
and normal skin following local injection of VEGF-A165 protein,32,33
ferritin extravasated from venules by a trans-endothelial cell pathway, that provided by the vesiculo-vacuolar organelle (VVO). VVOs are grape-like clusters of uncoated, largely para-junctional, cytoplasmic vesicles and vacuoles that traverse endothelial cytoplasm from lumen to ablumen (Figure 3)
.31-35
The individual vesicles and vacuoles that comprise VVOs are linked to each other, and to the luminal and abluminal plasma membranes, by stomata that are normally closed by thin diaphragms. By some as yet unknown mechanism, VEGF-A164/5 (and other vasoactive mediators) causes these stomata to open, providing a pathway for plasma and plasma protein extravasation. By contrast, interendothelial cell junctions remained tightly closed and did not admit tracer ferritin, even in the presence of abundant VEGF-A164/5.
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Within 18 hours of Ad-VEGF-A164 injection, enlarged, thin-walled, serpentine, pericyte-poor, hyperpermeable, and strongly VEGFR-2-positive sinusoids appeared, structures to which we have given the name "mother" vessels (Figure 4)
. Vessels of this description are commonly found in human and animal tumors (Figure 4k)
35
and are also transiently present in healing wounds (Figure 4l)
.36
They continued to develop for 
5 days following injection of Ad-VEGF-A164 into tissues.
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4-fold, mean vessel perimeter by 2-fold, and the percent of dermis occupied by microvessels by 4- to 7-fold.26
Mother vessels formed by a multistage process that involved proteolytic digestion of vascular basement membranes, pericyte detachment, and spreading and thinning of endothelial cells to cover a greatly expanded surface area (Figure 3b and Figure 4)
. For the first 48 hours, mother vessels formed without significant endothelial cell or pericyte division; thereafter, both types of cells proliferated extensively.
The vascular basement membrane is an especially important impediment to microvessel enlargement. Intact basement membranes are non-compliant (inelastic) structures that limit vascular enlargement to an 30% increase in cross-sectional area.37
Therefore, structural changes in the vascular basement membrane were necessary to accommodate the dramatic increases in microvessel area that were associated with VEGF-A164-induced mother vessel formation. VEGF-A is known to up-regulate the expression of endothelial cell proteases that degrade vascular basement membranes4,18
and reduced or absent staining for laminin and other basement membrane components, indicative of basement membrane degradation, was demonstrated by immunohistochemistry in developing mother vessels (Figure 4h)
.26
A Role for VVOs in Mother Vessel Formation
In addition to providing a pathway for macromolecule extravasation, VVOs also had a critical role in mother vessel formation. They provided an intracellular store of membrane that could be rapidly mobilized to the plasma membrane to increase cell surface area and permit vessel enlargement. Normal venular endothelium stores an amount of membrane in VVOs that is equivalent to 2.25 times that of plasma membrane.20
Therefore, transfer of VVO membranes to the cell surface has the potential to provide a >3-fold increase in endothelial cell perimeter without new membrane synthesis. Consistent with this possibility, mother vessel formation was accompanied by progressive endothelial cell thinning and reduction in VVO vesicles and vacuoles (Figure 3b
, unpublished data). Our findings, therefore, indicate that the endothelial cell spreading and thinning that accompany mother vessel formation can be accommodated, at least initially, by transfer of intracellular VVO membrane to the cell surface.
Mother Vessel Evolution
Mother vessels are transitional forms that evolved along several different pathways (Figure 5)
.26
Many underwent a process of bridging, in which cells expressing endothelial markers projected processes into and across mother vessel lumens (Figure 4, f, i, j)
. It remains to be determined whether these cells represent locally-activated vascular endothelium or were instead derived from circulating bone marrow precursors.38,39
Whatever their source, they formed transluminal bridges that divided blood flow into multiple smaller-sized channels. Similar bridging takes place in tumor mother vessels (Figure 4k)
, and we have postulated that at least some of the smaller channels separated from each other to form individual smaller-caliber daughter capillaries.35,40
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GB are poorly organized vascular structures that resemble renal glomeruli (hence the name) and are a common feature of several human tumors, among them glioblastoma multiforme.41
GB precursors were first recognized at 3 days as focal collections of large, primitive cells in the endothelial lining of mother vessels (Figures 5 and 6)
.42
These cells bore endothelial cell markers, proliferated rapidly, and extended both into the vascular lumen and outwards into the extravascular connective tissue. By 7 to 10 days, pericytes were prominently included and organized themselves around endothelial cells to form primitive microvessels. As they expanded, GB severely compromised the mother vessels in which they had arisen, reducing and dividing originally large single vascular lumens into multiple, smaller, tortuous channels.
After about 14 days, as VEGF-A164 expression declined, GB progressively devolved by a process that involved both apoptosis and reorganization of endothelial cells and pericytes to form normal appearing capillaries (Figure 6i)
. By 8 weeks, VEGF-A164 expression was no longer detectable and the angiogenic response had largely resolved with a return of microvascular density to near-normal levels. Taken together, these experiments provided the first animal model for generating GB, offered important insights into the mechanisms of GB formation and devolution, and demonstrated that VEGF-A164 is both sufficient for GB generation and necessary for their maintenance.
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Thin-walled mother vessels lacking pericyte and basement membrane support are subject to thrombosis or collapse. While many mother vessels avoid this fate by evolving into smaller daughter vessels or GB, others maintained their large size by acquiring a coat of smooth muscle cells and/or collagen-expressing fibroblasts.25
Such vessels were readily distinguished from normal arteries and veins by their inappropriately large size, by their thinner and often asymmetric muscular coat, and by the presence of perivascular fibrosis (Figure 7, a and b)
. Vessels of this description closely resemble those found in vascular malformations.43
This finding is of interest because it provides an animal model for generating vascular malformations and suggests that the vascular malformations observed in patients may be induced by VEGF-A overexpression. Once formed, these malformations persisted indefinitely (for more than a year), long after VEGF-A164 expression had ceased, indicating that they had achieved VEGF-A164 independence. In parallel with the formation of vascular malformations, small arteries and arterioles also responded to VEGF-A164 with modest replication of endothelial and smooth muscle cells, leading to the formation of larger, well-differentiated arteries (Figure 7, c–f)
.20
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Unexpectedly, VEGF-A164 also stimulated the proliferation of lymphatic endothelium, leading to the formation of a dense meshwork of tortuous, irregularly shaped, giant lymphatic channels (Figure 8)
.44
These giant lymphatics were functional but poorly so, clearing tracers over a period of many hours rather than in a few minutes as in normal lymphatics.
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3 days after injection of Ad-VEGF-A164, increased in size and number over the course of several weeks, and persisted indefinitely, in some instances for >1 year. Unlike venules, normal lymphatics lack pericytes and a well-developed basement membrane and are able to enlarge in response to edema.45,46
Therefore, formation of giant lymphatics was largely attributable to replication of lymphatic endothelial cells without the need for basement membrane degradation, pericyte detachment, or endothelial cell thinning as was necessary for the formation of mother vessels. Transformation of Provisional Fibrin Gel into Mature Connective Tissue Stroma
As was noted earlier, fibrin deposited in tumors and wounds imposes structure (Figure 1, a and b)
. It also provides an environment that supports cell migration (Figure 1, h–j)
. Endothelial cells, fibroblasts, and inflammatory cells express adhesion molecules (integrins) whose interaction with fibrin and fibronectin allows them to move freely in tumor and wound stroma.29,47
As a consequence, fibrin facilitates both angiogenesis and the inward migration of fibroblasts. Over time, as VEGF-A164 expression declined, vascular permeability returned to normally low levels and the balance shifted from fibrin accumulation to fibrinolysis. Eventually, fibrin was digested, small blood vessels underwent apoptosis, and fibroblasts synthesized collagen, resulting finally in dense, relatively avascular connective tissue.1,48
Commentary
A number of conclusions can be drawn from these studies. First, VEGF-A164 sets in motion a complicated chain of events that leads to the generation of a vascular and connective tissue stroma that closely resembles that found in animal and human tumors. Because nearly all malignant tumors express VEGF-A164/5, there is every reason to believe that tumors use this cytokine to generate stroma. Further, the steps and mechanisms by which tumors generate stroma closely mimic the events of wound healing in which VEGF-A164/5 is also expressed.36 Both tumor stroma generation and wound healing are characterized by vascular hyperpermeability, leakage of plasma, and clotting of extravasated fibrinogen to form a fibrin gel that serves as a provisional stroma for blood vessel and fibroblast migration. In both, the fibrin gel is degraded and the vascular connective tissue subsequently replaced by desmoplasia (tumors) or scar (wounds). However, whereas in healing wounds VEGF-A expression ceases with restoration of tissue normoxia, in tumors VEGF-A is expressed indefinitely. Thus, tumors may be likened to wounds that do not heal.1
Another important point is that the new blood vessels induced by Ad-VEGF-A164 and by tumors are not of a single kind but rather can be classified into at least several morphologically distinct types, ie, mother vessels, glomeruloid bodies, and vascular malformations. This heterogeneity may be expected to complicate anti-angiogenic and anti-vascular tumor therapy because not one but several distinct types of blood vessels must be targeted. Also, whereas mother vessels and glomeruloid bodies required continued VEGF-A164 expression for maintenance, vascular malformations did not and therefore would not be expected to respond to therapies that neutralized VEGF-A164 or its receptors. Consistent with this line of thinking, Benjamin et al23 have shown that withdrawal of VEGF-A leads to the selective apoptosis of only those tumor microvessels that lack a pericyte coating.
In addition to inducing angiogenesis (formation of small blood vessels) VEGF-A164 also induced vascular malformations and arteries. These large vessels could have clinical significance quite apart from cancer. Administration of VEGF-A164/5 and other angiogenic cytokines has been proposed as a novel approach for the treatment of coronary heart disease and other examples of tissue ischemia.49 However, the angiogenesis induced by VEGF-A164/5 would not be expected to offer much clinical benefit, first because the response is transient and second because the small vessels that form in response to VEGF-A supply a newly induced stroma, not the native tissue. Instead, relief of tissue ischemia requires large patent arterial blood vessels that are located proximal to, not within, the ischemic tissue. The vascular malformations and arteries induced by VEGF-A164 might satisfy this criterion because they are of appropriate size and persist indefinitely, independent of VEGF-A. It is possible, therefore, that some of the clinical benefits that have been reported for gene therapy with VEGF-A49 resulted not from angiogenesis but from unrecognized formation of these larger vessels.
The finding that VEGF-A induced lymphangiogenesis was unanticipated. Other members of the VEGF-A family, VEGF-C and VEGF-D, have been thought to be responsible for lymphatic development.50 However, we found no evidence for VEGF-C or VEGF-D expression in our studies of Ad-VEGF-A164, and it seems more likely that VEGF-A164 acted directly on lymphatic endothelial cells through VEGFR-2.51 Our findings predict that malignant tumors that express large amounts of VEGF-A164/5 would also induce the formation of abnormal lymphatics. Tumors have not been thought to generate new lymphatics52 but this issue needs to be reinvestigated in light of our findings.
In summary, our work has demonstrated that a single cytokine, VEGF-A164, induces remarkably diverse vascular responses and can account for at least three of the distinct types of abnormal blood vessels found in tumors. We do not yet know the secondary factors that are responsible for generating each of these vessel types and the larger number of such factors that must be needed to form the diverse types of mature blood vessels that are found in normal adult tissues. Discovering these secondary factors and elucidating their mode of action is an important future goal that will have important implications for understanding normal vascular development and for developing both pro- and anti-angiogenic therapies that have clinical utility.
Footnotes
Address reprint requests to Harold F. Dvorak, M.D., Department of Pathology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. E-mail: hdvorak{at}bidmc.harvard.edu
Supported in part by U.S. Public Health Service grants CA-50453, HL-59316, and P01 CA92644 and by a contract from the National Foundation for Cancer Research.
The Rous-Whipple Award was established by the American Society for Investigative Pathology to recognize a career of outstanding scientific contribution. Harold F. Dvorak, the 2002 recipient of the Rous-Whipple Award, delivered a lecture entitled, "How Tumors Make Bad Blood Vessels and Stroma" after accepting the award at the 2002 annual meeting of the American Society for Investigative Pathology in New Orleans, Louisiana.
Accepted for publication February 27, 2003.
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S.-H. Chang, C. H. Liu, R. Conway, D. K. Han, K. Nithipatikom, O. C. Trifan, T. F. Lane, and T. Hla From the Cover: Role of prostaglandin E2-dependent angiogenic switch in cyclooxygenase 2-induced breast cancer progression PNAS, January 13, 2004; 101(2): 591 - 596. [Abstract] [Full Text] [PDF]
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-smooth-muscle actin. Note incomplete pericyte covering, especially over segments of greatest vessel enlargement. c: Developing mother vessels illustrating pericyte detachment (arrows). d: Mother vessel with detached pericyte (arrow) and activated endothelial cells whose large nuclei bulge into the vascular lumen, creating a highly irregular surface. e and f: Serpentine mother vessels with irregular luminal surface and endothelial cell bridging (f, arrow). g: Mother vessels (m) embedded in fibrin (arrows) provisional stroma. h: Loss of laminin staining (arrows) in developing mother vessels (m). i and j: Endothelial cell bridging (i, arrows) in mother vessels in skeletal muscle and ear skin, respectively. k and l: Mother vessels in mouse ovarian tumor and healing rat skin wound, respectively. Note bridging in k (arrows) and fibrin in l (lower, between cells). Scale bars, 20 µm (b–l); 100 µm (a). a and b: Reprinted
