[Novartis Foundation Symposia] Ciba Foundation Symposium 100 - Development of the Vascular System...

Inhibition of neovascularization by a cartilage factor

KLAUS E. KUETTNERa,C and BENDICHT U. PAULIa,b

Departments of Biochemistrya, Pathologyb. and Orthopedic Surgeryc, Rush Medical College, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612, USA

Abstract. Neovascularization of developing, repairing or neoplastic tissues is regulated, at least partially, by a family of proteins of low molecular mass (1000<mol mass <50 000 Da) which can be extracted from avascular tissues, such as hyaline cartilage, aorta or bladder epithelium, by mild salt solutions. These extractable proteins, functionally defined as anti-invasion factor (AIF), act as local regulators for some of the major mechanistic pathways by which endothelial cells are thought to invade tissues during neovascularization, mainly by matrix-degrading enzymes and by increased rates of migration and proliferation. AIF contains a spectrum of proteinase (collagenase) inhibitory activities, as well as an endothelial cell growth inhibitor. The endothelial cell growth inhibitor is directed against actively dividing endothelial cells in culture but has no effect on endothelial cell monolayers or any other cell lines tested. In tumours, the AIF-derived endothelial cell growth inhibitor may limit tumour growth to less than 2 m m in diameter by inhibiting tumour neovascularization.

1983 Development of the vascular system. Pitman Books, London (Ciba Foundation symposium 100) p 163-173

In normal mammalian tissues, blood capillaries are embedded in a connective tissue matrix that consists of a meshwork of tightly packed and highly cross-linked collagen fibrils. These fibrils are embedded in a viscoelastic ground substance whose major components are structural macromolecular complexes, i.e. proteoglycans, glycoproteins and elastin (for review see Hay 1981). The normal packing of these macromolecules may leave little or no space for the budding and sprouting of capillary endothelia observed during neovascularization processes. However, activated endothelial cells may over- come such barriers physically through their own momentum (i.e. locomotion, proliferation), at the same time making full use of their capacity to adapt their shape. Alternatively, they can penetrate the barrier chemically by loosening or dissolving it with the help of matrix-degrading enzymes (proteinases). This enzymic lysis of extracellular matrices during endothelial cell invasion

163

Development of the vascular system Jonarhon Nugenr and Maeve OConnor

Copyrighr@ClBA FOUNDAnON

164 KUETTNER & PAUL1

(neovascularization) may not require the total destruction of tissue barriers. Lowering the level of molecular organization may be sufficient to reduce physical resistance to vascular invasion (Kuettner & Pauli 1982). For example, endothelial cells might break cross-links between collagen molecules or intermolecular bonds between various types of matrix macromolecules, causing swelling of glycosaminoglycan-containing proteoglycans and solu- bilization of the extracellular matrix (Pauli et a1 1983). This transient loosening of extracellular matrices in front of budding capillaries may facilitate the access of endothelial cell sprouts to deeper zones of developing, repairing or neoplastic tissues.

Endothelial cell invasion of tissues during neovascularization seems to be initiated by an angiogenic factor (i.e. tumour angiogenic factor, retinal extract, heparin) (Folkman & Cotran 1976, Azizkhan et a1 1980). These angiogenic factors may have rnitogenic activity or chemotactic activity, or both, in capillary endothelial cells (Fenselau et a1 1981). Furthermore, they may trigger the synthesis and secretion of proteinases by sprouting capillary endothelial cells. Indeed, bovine capillary endothelial cells stimulated in vitro with angiogenic factors (i.e. retinal extracts, conditioned medium from mouse adipocyte cultures, sonicates of cultured hepatoma cells), synthesize plasminogen activator and collagenase in a dose-dependent manner (Rifkin et a1 1981). Synthesis of plasminogen activator and collagenase is limited to capillary endothelial cells, the cells actually involved in neovascularization. It is not observed in endothelial cells derived from other sources, such as bovine aortas.

For most tissues, neovascularization is necessary to provide nutrients and to eliminate waste products. However, there are several tissues, such as cartilage, cornea, dentin, heart valves, lens and epithelia, which do not normally vascularize. In these tissues, avascularity seems to be essential for proper physiological function. The resistance of these tissues to endothelial cell invasion was first studied using the chick chorioallantoic membrane (CAM) as a biological assay system (Eisenstein et a1 1973, Sorgente et a1 1975, Kuettner et a1 1976b). With proper techniques, it is possible to observe neovascularization of grafted tissue on the immunologically protected CAM. Since avian erythrocytes are nucleated it is possible to determine, by simple histological studies, whether blood vessels found in mammalian grafts are of graft or host origin. Direct observations of the tissue grafts during the incubation period are made through a window placed in the egg shell.

Vascularized graft tissues are readily penetrated by the vessels of the CAM. In contrast, graft tissues such as hyaline cartilage or cornea that are devoid of any intrinsic blood supply are enveloped but never penetrated by the blood vessels of the CAM (Eisenstein et a1 1973). The hyaline cartilage of grafted rib costochondral junctions is never penetrated, even though it is eventually

INHIBITION OF NEOVASCULARIZATION 165

completely covered by a pannus of vascularized mesenchyme. Only the vascularized regions of the explant, such as bone tissue or calcified cartilage of the growth plate, are invaded by the blood vessels of the CAM. The chick blood vessels penetrate as far into the growth plate as the intrinsic vessels normally reach, namely to the area of the last hypertrophic chondrocyte and its adjacent calcified matrix, but they are stopped by the uncalcified columnar cartilage. This inhibition of invasion by vascular mesenchyme does not appear to be directly controlled by the activity of the chondrocytes, since it persists after cells of the graft are devitalized by freezing and thawing (Eisenstein et a1 1973, Kuettner & Pauli 1978).

Using the CAM assay system we have been able to document that cartilage contains diffusible or extractable substances which inhibit invasion of the cartilage matrix by capillary endothelial cells. Normal as well as physically or chemically altered cartilage pieces are placed on Millipore membranes and grafted onto the CAM. Bare filters are covered by arcades of blood vessels within six days of incubation. However, when cartilage explants are placed on the filters the vascular proliferation is markedly diminished, suggesting that a diffusible factor from the explant inhibits blood vessel proliferation (Kuettner et a1 1976b). This inhibitory effect on vascular proliferation is also evident when Millipore membranes carrying freeze-powdered cartilage are transplanted onto the CAM. Blood vessels and accompanying connective tissue elements, which normally cover the Millipore membrane, are unable to approach or penetrate the cartilage powder pellet. These observations are consistent with those described by Brem et a1 (1975) and Folkman & Cotran (1976). These investigators showed that cartilage pieces placed between a tumour and a potential vascular source in the anterior chamber of the rabbit eye inhibited the proliferation of limbal vessels that were otherwise stimulated to grow into the cornea by the tumour implant. Isolated chondrocytes, as well as cartilage extracts, that were injected intradermally also inhibited the angiogenic activity of lymphocytes during local graft-versus-host reactions (Kaminski et a1 1977).

These findings led us to postulate that cartilage contains factors that regulate the formation of new blood vessels during neovascularization of tissues. To substantiate this hypothesis, we extracted hyaline cartilage fragments mildly with guanidine hydrochloride, washed the extracts with physiological saline, then explanted them onto the CAM (Sorgente et a1 1975). Within seven days of incubation the extracted explants were covered with a dense vascularized mesenchyme and were invaded by numerous capillary branches (Kuettner & Pauli 1978). We therefore extracted hyaline cartilage on a large scale, in order to analyse the extractable substances for their effects on the invasive apparatus of vascular endothelial cells.

The isolation procedure for the cartilage-derived anti-invasion factor (AIF)


is standard and has been described in detail (Eisenstein et a1 1975, Sorgente et a1 1975, Kuettner et a1 1977, Horton et a1 1978). Slices of fresh hyaline cartilage, prepared from the nasal septa of 18-month-old cows, were extracted with 1 M-NaCl (0.05 M-sodium acetate, pH 5.8, 24h, 4°C). We decanted the extract from the tissue and adjusted the preparation to 3M-NaC1 by adding solid NaCl to minimize non-specific protein-protein interactions. Ultrafiltration of the crude cartilage extracts yielded two fractions: (1) the XM-50 retentate (>50000Da), and (2), after dialysis and concentration, the UM-2 retentate designated AIF (1000 < mol mass < 50 000 Da). The XM-50 retentate contained most of the proteins, as indicated by standard biochemical analysis (uronic acid, hexose, hydroxyproline), In contrast, AIF contained only about 4Opg protein/g tissue, with minimal amounts of uronic acid, hexose and hydroxyproline. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that AIF consisted of seven major protein bands. The protein with the highest molecular mass co-migrated with serum albumin (69 000 Da). Immunologically identified albumin was present in this preparation, due to its incomplete rejection by the XM-50 membrane, as described by the manufacturer (Amicon Corp.). The protein with the lowest molecular mass migrated between TrasylolB (6500 Da) and insulin (5700 Da).

AIF expresses inhibitory activity against a variety of proteinases. Originally we showed (Kuettner et a1 1976a, 1977) that AIF contains a cationic protein fraction which possesses inhibitory activity directed against both trypsin and mammalian collagenases. The inhibitory activities affecting both trypsin and collagenase eluted from an insoluble trypsin affinity column were due to a single protein of approximately 11 000 Da. However, since then Roughley et a1 (1978) have shown that the inhibition of trypsin and mammalian collagenases resides in distinct molecules, and that bovine nasal cartilage contains a third inhibitor that is directed against thiol proteinases such as cathepsin B and papain. By gel chromatography, the inhibitors of collagenases, thiol proteinases and trypsin are eluted with apparent molecular masses of about 22 000, 13 000 and 7000 Da, respectively. The trypsin inhibitor appears at about the same elution volume as the commercially available basic pancreatic trypsin inhibitor, TrasylolB (aprotinin). The trypsin inhibitor resembles Trasylol in its molecular mass, antigenicity and range of susceptible proteinases but it shows slight variations in amino acid composition (Rifkin & Crowe 1977). Susceptible proteinases are trypsin, chymotrypsin, plasmin and proteoglycan-degrading enzymes derived from human leucocyte extract, and neutral proteinases associated with the tumour cell surface (for review see Pauli & Kuettner 1982). Cartilage-derived AIF also expresses inhibitory activities against neutral metalloproteinases that cleave non-interstitial collagen types IV and V (Pauli & Kuettner 1982) and against human neutrophil


elastase (Kuettner & Pauli 1982). The spectrum of proteinase inhibitors present in extracts of bovine hyaline cartilage may play a significant role in the regulation of neovascularization. The inhibition of major classes of matrix- degrading enzymes might prevent destruction and penetration of connective tissue barriers by sprouting capillary endothelia (Kuettner & Pauli 1982).

Cartilage-derived AIF has a strongly inhibitory effect on endothelial cell growth (Pauli & Kuettner 1982, Kuettner & Pauli 1982). When the double- labelling technique of Eckel & Fujimoto (1981) was used, AIF caused [“Clthymidine to be lost from prelabelled bovine aortic endothelial cells in a dose-dependent manner. Loss of label was due to the lethal effect of AIF on actively dividing endothelial cells. Exposed cells became spherical after 2-4 h, sloughed in large numbers from the plastic growth surface, and died. Simultaneous measurements of [3H]thymidine incorporation into the [14C] thymidine-prelabelled endothelial cell cultures indicated that the remaining endothelial cells proliferated at a slower rate than those of unexposed control cultures. At an optimal concentration of 5Opg proteiniml culture medium, and after an exposure time of 24 h, AIF caused a loss of more than 50% of endothelial cells and reduced the growth rate of the remaining viable endothelial cells by about 40%. AIF protein concentrations below l p g proteidml culture had no effect on actively dividing endothelial cells. AIF had no significant effect on endothelial cells of stationary monolayers at any of the concentrations tested. Control cultures of fibroblasts from normal human foreskin or epithelial cells from the normal rat urinary bladder were not affected by AIF at any time during their growth in culture. The AIF-derived growth factor was also ineffective against endothelial cells that had been carried in culture for more than 12 passages and had lost the ability to synthesize factor VIII.

Sorgente & Dorey (1980) have isolated and partially purified the endothelial cell growth inhibitor from an extract of bovine scapular cartilage. The endothelial cell growth inhibitor is distinct from the proteinase inhibitors and is non-cytotoxic. It increases the doubling time of endothelial cells from 24 h to 40 h. These findings suggest that the growth inhibitor arrests endothelial cells in the GI phase. The endothelial cell growth inhibitor derived from hyaline cartilage seems to be identical to the anti-angiogenic activity described by Brem et al (1975) and Folkman & Cotran (1976). Using the rabbit cornea as an assay system, these investigators showed that neovascularization of tumours could be inhibited when a small piece of viable hyaline cartilage was positioned between a tumour implant and the corneoscleral junction.

Similar factors have been prepared from extracts of other avascular tissues, that is from bovine aorta and bovine urinary bladder mucosa. The bladder- derived AIF contained (1) a Trasylol-like trypsin inhibitor, (2) a collagenase


inhibitor, and (3) an endothelial cell growth inhibitor (Waxler et a1 1982). The endothelial cell growth inhibitor had a lethal effect on actively dividing vascular endothelial cells, as shown by the loss of [14C]thymidine from prelabelled endothelial cell cultures. Fibroblasts, normal bladder epithelial cells and a variety of human and animal tumour cell lines were not affected by the bladder-derived AIF at any time during their growth in culture. By inhibiting basement membrane-degrading enzymes and endothelial cell proliferation, this factor may effectively inhibit the vascularization of bladder epithelium and thus secure proper epithelial function by maintaining osmotic and ionic gradients between urine and blood.

Extracts from bovine aorta contained at least four separate molecular species capable of affecting both invasive and proliferative processes (Eisen- stein et a1 1979a, b). Two of these molecular species were large anions, namely heparin and a dermatan sulphate-chondroitin sulphate-containing proteoglycan. These structural macromolecules inhibited the growth of both smooth muscle cells and endothelial cells, but large amounts were required to induce this effect (Eisenstein et a1 1979b). The other two molecular species were part of the aortic extract fraction of low molecular mass. The first was a proteinase inhibitor which resembled the basic pancreatic trypsin inhibitor, Trasylol. The second was a potent inhibitor of endothelial cell growth that had a molecular mass between 3500 and 10000Da. In the rabbit eye, the aortic extract fraction of low molecular mass, administered either subcon- junctivally or topically, inhibited corneal oedema and neovascularization after silver nitrate cautery (Eisenstein et a1 1979a). Because subconjunctival injections as late as 48 h after cautery were effective, it was concluded that inhibition of corneal oedema and neovascularization by bovine aortic extracts were not entirely caused by interference with the initial stimulus to neovascularization generated by the injury. The finding that commercially supplied Trasylol had no effect on corneal oedema and neovascularization after silver nitrate cautery indicated that the effective molecule in the aortic extract might be the endothelial cell growth inhibitor. This hypothesis was supported by the finding that the growth of endothelial cells in culture was inhibited. At a concentration of 100,ugiml the aortic extract inhibited endothelial cell growth by 60% (Eisenstein et a1 1979a). Growth inhibitory activity in aortic extracts, like that seen with cartilage AIF, was effective only on actively dividing endothelial cells and had no effect on endothelial cell monolayers.

In conclusion, the resistance of cartilage (and other avascular tissues) to vascular invasion may be due to a family of extractable proteins of low molecular mass, functionally defined as anti-invasion factor. This factor can be extracted with mild salt solutions from uncalcified hyaline cartilage, aorta and urinary bladder mucosa, or, as shown only recently, from cultures of bovine articular chondrocytes. The biological activities of the salt extract,


such as inhibition of proteinase and of endothelial cell growth, are present in a protein fraction of between 1000 and 50 000 Da. These activities seem to act as local regulators for some of the major pathways by which endothelial cells are thought to invade developing, repairing or neoplastic tissues, namely by matrix-degrading enzymes and by increased rates of proliferation and migration. In tumours these inhibitors may limit tumour growth to less than 2mm in diameter by inhibiting tumour neovascularization (Pauli & Kuettner 1982). The small molecular masses of the inhibitors allow relatively free access to tissue sites, thus increasing their efficacy.

Acknowledgements

We wish to thank our research staffs for having contributed so much of the information in this review, and Ms C. H . Sanes-Miller for her editorial assistance. This work was supported by Public Health Service (PHS) Grant CA-21566 from the National Cancer Institute (NCI), and in part by PHS Grant CA-25034 from the NCI and by Grants R-1206 and R-1394 from the Council for Tobacco Research-USA, Inc.

REFERENCES

Azizkhan R G , Azizkhan JC, Zetter B R , Folkman J 1980 Mast cell heparin stimulates migration of capillary endothelial cells in vitro. J Exp Med 152:931-944

Brem H , Arensman R , Folkman J 1975 Inhibition of tumor angiogenesis by a diffusable factor from cartilage. In: Slavkin HC, Greulich R C (eds) Extracellular matrix influences on gene expression. Academic Press, New York, p 767-772

Eckel R H , Fujimoto WY 1981 Quantification of cell death in human fibroblasts by measuring the loss of [14C]-thymidine from prelabeled cell monolayers. Anal Biochem 114:118-124

Eisenstein R . Sorgente N, Soble LW. Miller A, Kuettner KE 1973 The resistance of certain tissues to invasion. I. Permeability of explanted tissues by vascularized mesenchyme. Am J Pathol 73~765-774

Eisenstein R. Kuettner KE, Neapolitan C, Soble LW, Sorgente N 1975 The resistance of certain tissues to invasion. 111. Cartilage extracts inhibit growth of fibroblasts and endothelial cells in culture. Am J Pathol 81:337-348

Eisenstein R , Goren SB, Schumacher B , Choromokos E 1979a The inhibition of corneal vascularization with aortic extracts in rabbits. Am J Ophthalmol 88:1005-1012

Eisenstein R , Harper E, Kuettner KE. Schumacher B, Matijevitch B 1979b Growth regulators in connective tissues. 11. Evidence for the presence of several growth inhibitors in aortic extracts. Paroi Arttrielle 5:163-169

Fenselau A , Watt S, Mello RJ 1981 Tumor angiogenic factor. J Biol Chem 256:9605-9611 Folkman J , Cotran R 1976 Relation of vascular proliferation to tumor growth. Int Rev Exp

Hay E D (ed) 1981 Cell biology of extracellular matrix. Plenum Press, New York Horton J E , Wezeman F H , Kuettner KE 1978 Inhibition of bone resorption in vitro by a

cartilage-derived anti-collagenase factor. Science (Wash DC) 199: 1342-1345

Pathol 16:207-248

170 DISCUSSION

Kaminski M, Kaminska G , Jakobisiak, M, Brzezinski W 1977 Inhibition of lymphocyte-induced angiogenesis by isolated chondrocytes. Nature (Lond) 268:238-240

Kuettner KE, Pauli BU 1978 Resistance of cartilage to normal and neoplastic invasion. In: Horton JE et a1 (eds) Mechanisms of localized bone loss. Information Retrieval, Washington, DC (special supplement, Calcif Tissue Abstr) p 251-278

Kuettner KE, Pauli BU 1982 Vascularity of cartilage. In: Hall BK (ed) Cartilage. Academic Press, New York, 1:281-312

Kuettner KE, Hiti J , Eisenstein R, Harper E 1976a Collagenase inhibition by cationic proteins derived from cartilage and aorta. Biochem Biophys Res Commun 72:40-46

Kuettner KE, Soble LW, Sorgente N, Eisenstein R 1976b The possible role of protease inhibitors in cartilage metabolism. In: Peeters H (ed) Protides of the biological fluids. Pergamon Press, Oxford (Proc 23rd Colloq Protides Biol Fluids) p 221-225

Kuettner KE, Soble L, Croxen RL, Marczynska B, Hiti J , Harper E 1977 Tumor cell collagenase and its inhibition by cartilage-derived protease inhibitor. Science (Wash DC) 196:653-654

Pauli BU, Kuettner KE 1982 The regulation of invasion by a cartilage-derived anti-invasion factor. In: Liotta LA, Hart IR (eds) Tumor invasion and metastasis. Martinus Nijhoff, The Hague, p 267-290

Pauli BU, Schwartz DE. Thonar EJ-M, Kuettner KE 1983 Tumor invasion and host extracellular matrix. Cancer Metastasis Rev, in press

Rifkin DB, Crowe RM 1977 Isolation of protease inhibitor from tissues resistant to tumor invasion. Hoppe-Seyler’s Z Physiol Chem 358: 1525-1.531

Ritkin DB, Gross JL, Moscatelli D, Gabrielides C 1981 The involvement of proteases and protease inhibitors in neovascularization. Acta Biol Med Ger 40:1259-1263

Roughley PJ, Murphy G , Barrett AJ 1978 Proteinase inhibitors of bovine nasal cartilage. Biochem J 169:721-724

Sorgente N, Dorey CK 1980 Inhibition of endothelial cell growth by a factor isolated from cartilage. Exp Cell Res 128:63-71

Sorgente N, Kuettner KE, Soble LW, Eisenstein R 1975 The resistance of certain tissues to invasion. 11. Evidence for extractable factors in cartilage which inhibit invasion by vascularized mesenchyme. Lab Invest 32:217-222

Waxler B, Kuettner KE. Pauli BU 1982 The resistance of epithelia to vascularization: proteinase and endothelial cell growth inhibitory activities in urinary bladder epithelium. Tissue Cell 14 :657-667

DISCUSSION

Gluser: Do other avascular tissues such as the cornea and the vitreous show similar results in response to purified anti-invasion factor (AIF)?

Kuettner: We haven’t done the last steps of purification. A polyclonal antibody prepared against cartilage AIF cross-reacts with some of the proteins in the urinary bladder, but we haven’t gone any further with the vitreous or the cornea. We know that the cornea has a material with similar inhibitory activity.

Reynolds: But do you know that fractions that are active have anti-protease activity?


Kuetfner: We have so far isolated only a small quantity of material which inhibits endothelial cell growth. In this material we could not find anti- protease activity. I think we have separated the endothelial cell growth inhibitory activity from the proteinase inhibitory activity.

Gordon: When you talk about endothelial cell inhibitory activity, what precisely does that term mean?

Kueffner: As I described in my paper, we prelabel endothelial cells in culture with [14C]thymidine before adding AIF. Subcultures are also exposed to [3H]thymidine. Release of [14C]thymidine gives an estimate of cell loss. Inhibition of cell proliferation can be determined from the incorporation of [3H]thymidine into actively dividing endothelial cells, after adjustment for cell death.

Gordon: Did you say that the fraction with elastase inhibitory activity was the most powerful inhibitor of endothelial proliferation?

Kueffner: In the invasion chamber system, heparin-stimulated endothelial cells are able to penetrate into the extracted cartilage matrix. This process can be inhibited when the matrix is precoated with AIF, which contains inhibitors including an elastase inhibitor.

Gordon: Which fraction was the most powerful inhibitor of invasion? Kuetfner: We don’t really know that yet. Trasylol does not seem to be

effective. Most of the protein material has a molecular mass of less than 50 000 Da. Poswillo: Reconstructive surgeons have known for a long time that cartil-

age transplanted to vascular sites won’t resorb. Lyophilized cartilage, however, behaves in exactly the same way. Are these anti-invasion factors present in the lyophilized tissue?

Kuetfner: Yes, from our original experiments using the chorioallantoic membrane we could see an inhibitory effect with cartilage powder, which was lyophilized. What happens with implanted cartilage powder is not known. I suspect that the biologically active material will be exhausted if the chondrocytes cannot resynthesize it. We have some circumstantial evidence. If cartilage explants are grown on supporting grids without a Millipore membrane, osteosarcoma cells are unable to form a cell layer beneath these grids. This indicates that the cartilage has released, or probably synthesized, material that influences the growth of these tumour cells. Whether the tumour cells influence the chondrocytes has still to be investigated. Poswillo: This may explain one of the phenomena. In a 15-year study of

living cartilage and lyophilized cartilage the only significant difference is that after five or ten years the lyophilized cartilage begins to calcify (Sailer 1983). So the answer is that the inhibitory factor is wasted.

Folkman: The way those endothelial cells invaded the cartilage is fascinat- ing. That could be an analogue model of any matrix with the endothelial cell

172 DISCUSSION

sitting in its resting venule, then receiving some signal to go out, amplified by heparin. How long did that take?

Kuettner: I don’t know. The first observation was made after 48 hours. It is not a very fast process.

Folkman: Is it related to arthritis? Kuettner: There is a lot of interplay in degenerative arthritis. The process is

initially a self-destruction of the cartilage, not so much a destruction by outside forces.

Wolpert: I was going to ask you about the two different sides of the growth plate. Did it make a difference where you took the plug of tissue from?

Kuettner: Hyaline cartilage is never invaded. We wanted to use a real negative control, a cartilage which is never invaded by blood vessels under physiological conditions. Even in arthritic diseases there has to be massive destruction of the total cartilage before blood vessels invade. This is not true in rheumatoid arthritis, but in osteoarthritis and degenerative arthritis the cartilage matrix is significantly altered before the first blood vessels grow in.

Wolpert: And you say that in the growth plate the difference between the two sides is due to the difference in the distribution of your factor?

Kuettner: I can’t answer that yet. On the calcified side of the growth plate the blood vessels invade easily. If you inhibit calcification, vascular invasion ceases. In rachitic animals the growth plate can double in size but the blood vessels don’t invade since the matrix is not calcified. If we give vitamin D , calcification occurs. This is a good model for vascular sprouting. Within 24 hours the blood vessels recover the loss from several days of treatment. In another system of rickets induced by diphosphonates, the crystals are poisoned and vascular invasion also stops. Calcium seems to play a specific role in vascular invasion.

L e Douarin: Did you say that when you put a piece of cartilage from the dog onto the chick chorioallantoic membrane you got chicken bone cells? Do you mean that the (osteocyte) progenitors originate from the blood?

Kuettner: We know from the work of Kahn & Simmons (1975) that at least the osteoclasts are derived from the circulating blood cells.

L e Douarin: From our experience, that would not be the case (see Jotereau & Le Douarin 1978). Interspecific grafts of limb buds or of the embryonic cartilaginous femur on the chorioallantoic membrane have been done between quail and chick embryos. By following the cell lineage in the bone and marrow through the quail nuclear marker, we were able to show that the haemopoietic and the osteogenic (comprising osteoblasts, osteocytes and chondrocytes) cell lines have different embryological origins. The osteogenic line is derived from the limb bud mesenchyme or from the perichondrium lining the cartilaginous femur, while the haemopoietic cells are brought into the bone marrow via the circulation. In the fixed cells of the marrow two


categories have to be distinguished: the reticular cells originating from the bone rudiment and the endothelial cells which invade the cartilage and are of haematogenous origin. The osteoclasts belong to the haemopoietic cell line and are not derived from any cell type of the osteogenic line.

Young: There is a strong association between congenital anomalies of the vascular system and skeletal anomalies. A gross example is Maffucci’s syndrome where vascular anomalies are associated with dysplastic cartilage growth and occasionally even chondrosarcomas. A piece of cartilage from somebody with that syndrome might tell you something. Denekamp: Do you mean that the anomalies are anatomically close

together? Young: Frequently, but not necessarily.

REFERENCES

Kahn AJ, Simmons DJ 1975 Investigation of cell lineage in bone using a chimaera of chick and quail embryonic tissue. Nature (Lond) 258:325-327

Jotereau FV, Le Douarin NM 1978 The developmental relationship between osteocytes and osteoclasts: a study using the quail-chick nuclear marker in endochondral ossification. Dev Biol 63:253-265

Sailer H 1983 Transplantation of lyophilised cartilage in maxillofacial surgery: experimental foundation and clinical success. Karger, Bade (Monograph Series)

General discussion I1

Tumour angiogenesis factors

Reinhold: My understanding has been for a long time that neovascularization of tumours is a kind of reaction to metabolic demands. In our capillary proliferation system (Reinhold & Buisman 1973) we used the simplistic idea that in the absence of oxygen the cells would switch to anaerobic metabolism and make lactic acid. That would cause the capillaries to proliferate in the direction of the anaerobic cells. Dr Folkman, how important do you think the tumour angiogenesis factor (TAF), including its diffusion through the tumour tissue, is as the sole agent for angiogenesis in tumours?

Folkman: Our idea 10 years ago was that one would purify TAF, learn how to block it, and then turn off angiogenesis and possibly inhibit tumour growth. It has been more difficult to purify TAF and to block its activity than we had thought, although Dr Kumar’s group seems to have come closest. In fact, the discovery of several angiogenesis inhibitors that do not depend on inhibition of TAF itself has preceded purification of TAF. Also the purification of an angiogenic factor from normal tissue, namely the retina, by Glaser and D’A- more, is much further ahead than purification of tumour angiogenesis activity (Glaser et a1 1980). The chorioallantoic membrane, the rabbit cornea, and endothelial cell culture are the assays used by most workers.

The published work on angiogenic factors from tumours includes early work on chromatin, non-histone protein fractions of tumours and angiogenic factors of large and small molecular weight. Shant Kumar’s group has also reported an antibody to a tumour angiogenic factor derived from Walker 256 rat carcinosar- coma. The reports of angiogenic fractions of both high and low molecular mass may indicate the existence of a carrier protein, but it is too early to say what these reports mean. For angiogenesis inhibitors there is far more information and the field is moving faster than for angiogenic stimulators.

Wolpert: There seem to be two quite different culture assays. In one, the substance is added to the culture and the effect on proliferation is measured. But that doesn’t necessarily correlate very strongly with the other assay, based on the chorioallantoic membrane. Is that because the CAM assay is more complex since it is also measuring migration? In other words, proliferation is part of the mechanism but unless you have the total system you don’t actually get angiogenesis. Is that a way to look at it?

174

TUMOUR ANGIOGENESIS FACTORS 175

Folkman: That is one reason. It is the same problem as asking what causes coagulation of blood. Factor VIII or factor X alone can’t induce coagulation unless all the other factors are present and in the proper sequence. In angiogenesis, proliferation of endothelial cells is necessary but not sufficient.

Ross: An important caveat here applies to any factor one wants to purify to understand its biological significance. That is, one has to go through a series of stages. You need an in vitro assay simply because all in vivo assays take so long and are so complicated that you get nowhere in the initial screening process. When you have a relatively rapid screening method you can see and measure certain biological activities in vitro. The next question is, what does this have to do with anything in real life? Then you have to find an in vivo assay, but what works in vitro doesn’t seem to work in vivo, for a whole host of reasons. That doesn’t mean it does not work in vivo-i t means that you don’t have all the pieces of the puzzle. One then has to seek the other pieces of the puzzle because ultimately nobody will be really interested in what is happening in culture alone. The fact that TAF doesn’t work in the CAM means there are still pieces of the puzzle missing and they have to be put together. If it doesn’t work when all the other pieces are there, then you will have to re-think the problem.

Folkman: An example of Dr Ross’s caveat is that the tumour cell fractions that we used in migration assays increased migration, but often didn’t correlate with angiogenesis in the CAM. Inhibitors of migration in vitro correlated well with inhibitors of angiogenesis. On the other hand, tumour fractions that could stimulate capillary endothelial proliferation usually could stimulate angiogenesis in vivo. But not all mitogens for endothelial cells (in vitro) are angiogenic in vivo.

Wolpert: What is the evidence that something stimulating proliferation of endothelial cells has anything to do with angiogenesis?

Folkman: None. One must always eventually check with an in vivo assay. Wolpert: From the experiment you told us about earlier, in which

angiogenesis was stimulated in an irradiated eye, we learnt that cell proliferation is not essential to get angiogenesis. What, then, is the evidence that proliferation, or something that stimulates proliferation of endothelial cells, is necessary for angiogenesis?

Ross: Judah Folkman didn’t say that cell proliferation isn’t needed for angiogenesis. He said you don’t need cell proliferation for angiogenesis for the first two days and then further vessel migration stops. For angiogenesis you do need cell proliferation.

Folkman: It is possible to have an angiogenic factor that is chemotactic to endothelial cells and also stimulates proliferation, or it could be that there is at least one factor for each function.

176 GENERAL DISCUSSION 11

Roach: No one has talked about the flow in these vessels. If there is no flow in the vessels, what are they there for? It seems to me that you are starting back to front and that you should try to see whether the flow in tumour vessels is different from the flow in non-tumour vessels. Then you could decide where to look for vessels that will form new vessels, either by neovascularization or by sprouting. This is easy to do with some of the new techniques, particularly for things that grow in a relatively flat form that you can transilluminate.

Folkman: I agree. One can see flow in new vessels in the rabbit ear chamber or in the cornea. But I don’t know if anyone has made measurements.

Kumar: Some years ago we used pure and impure tumour-derived material and tested for angiogenesis. The impure material induced proliferation and migration of capillary endothelial cells. When we purified it, it induced proliferation but not migration of capillary endothelial cells and it had no effect whatsoever on aortic endothelial cells.

Fraser: Is the pure material from solid tumours or from tumour cell surfaces? Kurnar: From the solid tumour extract. Fraser: Presumably the material secreted by the tumour cells which is re-

coverable from their surface and to which the host cells are responding is not the purified compound. If the effects of tumour angiogenesis factor are being studied, then surely the naturally derived material, no matter how impure, should be used.

Le Douarin: Dr Folkman, you used two different assays in vivo, the rabbit eye and the CAM. Did you get parallel results with these two?

Folkman: In general, yes. The cornea assay permits better quantitation but is tedious and time-consuming and late immune reactions against the test substance may be confusing.

Le Douarin: Usually the tissue extracts come from mammalian tissues. Most histogenetic processes correlate very well between avian and mammalian species but others do not. The stroma of the thymus from one species of bird can be combined with haemopoietic cells from another avian species. But one can’t get the same result by combining the stroma of a mammalian thymus (mouse, for example) with haemopoietic cells from birds. Avian cells do not invade the stroma of the mammalian thymus, and vice versa. When you screen molecules extracted from mammalian tissue for their possible angiogenetic potency by using an assay that includes avian blood vessels, you may therefore miss something.

Folkman: That is a good point. When the CAM assay was first used, human and rat tumours grew well in it. We assumed then that the chick embryo, in terms of angiogenesis, could respond to human and rat tissues.

Auerbach: So far in our work the chick CAM assay, the intracorneal graft in mice and the intradermal graft in mice have always correlated, though there may be quantitative differences which we don’t measure in any of these


systems. Dr Le Douarin has shown that the endothelial cells share properties with the stem cells. What needs to be kept in mind when we measure how a tumour is vascularized in the chick CAM is not whether vessels are induced but what happens once the vessels get to the tumour. One can look at that, up to a point.

Ross: When you see angiogenesis does it bother you that you don’t see division in any of the endothelial cells? They have to be fully stretched out before cell division occurs somewhere further back. Have you ever created loops and added growth factors that stimulate capillary endothelial cells to multiply in cell culture, to see if there is some restriction on the cells in the loops that doesn’t permit DNA synthesis and cell division? Is there something different about the phenotypic state of the leading cells compared with the phenotypic state of the cells back at the base, that prevents them from responding?

Auerbach: There is an inaccuracy here which we need to clear up. When you look at vascular development in the eye, is it not true that once you get the sprouts you then get proliferation at the growing tips as well? If growth factors stimulate this, there will be tremendous differences in the number of new blood vessels that form. Tumours need not only to induce blood vessels but to induce lots of them.

Ross: But Judah Folkman says that is not what happened. Auerbach: That is what people have described in the published work. Folkman: Dianna Ausprunk has examined vascularized corneas by electron

microscopic autoradiography. The leading cells at the tip of the capillary are usually non-mitotic. But just behind them one sees the proliferating cells.

Ross: So you are actually getting multiplication very close to the edge of the tip?

Folkman: Yes. Gordon: If a vessel does not form a new branch by endothelial cell division,

how does it do it? Wolpert: The irradiation experiment shows that one can get a capillary

without any cell multiplication. Gordon: ‘Angiogenesis’ should mean the starting of a new blood vessel. One

reason for possible confusion in this discussion could be that angiogenesis is being used by some people to mean endothelial sprouting, whereas neovascularization (i.e. the formation of complete new vessels) requires proliferation as well. I would like to see a stricter definition of the term angiogenesis.

Wolpert: It is worse than that. Nothing that anybody has told me at this meeting suggests that the factors that stimulate proliferation of cells in culture have anything to do with what is happening in vivo.

Denekamp: Are you suggesting that angiogenesis is the production of capillary sprouts, and neovascularization is the production of extra endothelial

178 GENERAL DISCUSSION I1

cells? I think these two words are used interchangeably, but we do need to make a clear distinction between the formation of new vessels (branches) and the elongation of existing ones. Both these processes will lead to an increase in the overall vascular volume but the distribution and quality of the blood supply will differ in the two situations.

Gordon: I introduced this because Lewis Wolpert made the point that endothelial proliferation, as far as he understood it from the evidence of Judah Folkman and others, was not necessary for angiogenesis. Perhaps we need to clarify what is meant by angiogenesis: does it mean the first events that can be detected in the formation of new vessels, or does it mean the formation of a complete vascular network?

Ross: The evidence you mention comes from the radiation experiments that Judah has described. Those experiments have a lot of holes in them.

Wolpert: On the contrary, what Judah Folkman described is typical of several embryonic processes. Cell division usually only provides more cells, it does not organize the overall form of those cells. The morphogenetic process is quite separate. For example, it used to be thought that there was a growth at the base of the bud in Hydra and this caused the bud to grow out. But growth has nothing to do with it. If you give Hydra 20 000 rad they drop dead in two days but they bud first. Budding involves cell movement. It seems that it is the same for the vascular system. John Gordon’s point is very important. The crucial thing is the formation of the new vessel, the sprout. 1 believe proliferation is a totally separate thing.

Denekamp: A sprout is not a vessel, and the irradiation experiment described by Judah Folkman shows only that a limited expansion of the vascular network can occur without cell proliferation.

Ross: We are really talking about the formation of new blood vessels. The initial forming of a loop that Judah described is not the formation of a new blood vessel. It is only the beginning of the process. The formation of cells and morphogenesis are both part of the process, aren’t they?

Wolpert: I wouldn’t accept that. How much vascularization do you get after two days?

Folkman: Sholley et a1 (1980) saw new capillaries of 0.55 mm length at four days in rat corneas irradiated with 8000 rad. There was virtually no endothelial cell proliferation. The first loops had developed and there was flow. The opposite eyes were not irradiated and capillaries were 1.05 mm at four days. You can see flow. Sholley’s experiment shows that branching, lumen formation and migration are independent of cell division.

Denekamp: Is elongation of existing vessels going to count as angiogenesis? The vessels are already there in small tumours and cell proliferation may simply make them longer, bigger and wider, without creating any new branches.

Wolpert: That is what John Gordon would call vascularization.


Denekamp: I don’t understand his distinction. Tumours certainly grow by having longer vessels as well as by having an increase in the network.

Gordon: I am looking for clarification because there was confusion in the discussion, We have heard about different assay systems, some of which can identify endothelial proliferation, some of which can identify migration of endothelium or sprouting, and some of which are based on the formation of a complete new vascular bed. Neovascularization (the creation of complete new vessels, as I interpret the word) is a multiphasic process. It involves not only several endothelial functions but also smooth muscle and other cells. Endothe- lial cell proliferation is only one aspect of the process, and endothelial cell migration is another. My main aim in suggesting that we might clarify the terminology was to avoid the sort of confusion which we have already had when people haven’t made it clear which they meant. The term angiogenesis may mean different things to different people; can the experts present here agree on definitions so that terms such as angiogenesis can be applied more specifically in future?

Glaser: I think everybody is interested in finding out how to get from nothing to a new vascular bed, as happens in the cornea. It is probably a multi-step process. One of the steps might involve degradation of the extracellular matrix, the next step might be migration, and so on. There could be an enormous number of steps, including many we don‘t yet know about. The problem is that we don’t know enough about the steps. As we learn more about the steps involved in neovascularization we shall need to determine at which points they are controlled. Is something that stimulates endothelial cells to break down extracellular matrix components then needed to stimulate migration and proliferation, or once that first step has been taken can the other steps then proceed even in the absence of the initial stimulant? The experiment that we have been talking about says that if the cells cannot proliferate, vessels will still form, but we don’t know yet how the proliferation step fits in.

Gordon: When I stirred up this hornet’s nest about terminology I hoped we might agree on a term to describe the initiating step in new vessel formation and another term that would indicate the formation of a complete new vascular bed. However, there is apparently no general agreement on the nature of the initial step(s). I would therefore suggest that we don’t attempt, at present, to make precise definitions. But I hope we could agree that if angiogenesis (or any other word) is used to mean the formation of new vessels, then the term covers many separate but interrelated processes. It is therefore essential that it be made clear when only one particular process is being studied. For example, Shant Kumar referred to the properties of an impure and pure angiogenesis factor, saying that the impure factor affected both migration and proliferation of endothelium, while the pure factor affected only one of these processes. It is clear that his impure preparation contains at least two different activities, both


of which are relevant to new vessel formation; therefore, both (or all) the constituents responsible could justifiably be called angiogenesis factors. This illustrates the problem we have been discussing.

Denekamp: I still don’t know whether you are proposing to restrict the term angiogenesis to the development of new branches of the vasculature or whether you agree to include the elongation of existing branches. These both represent an expansion of the vascular network. As a tumour biologist I think that the budding inside a vascularized tumour is much less important than appears from the particular system Judah Folkman uses. He is providing a unilateral stimulus from the tumour fragment to the surrounding vessel, both in the eye and in the egg. However, once the tumour has incorporated its first vessels I am not convinced that this budding process is as prevalent as we have been making out.

This may also be true of some of the other vascular anomalies. Perhaps there is spiralling and an increase in the amount of vasculature but not necessarily any extra vessel branches. If we are going to restrict angiogenesis to mean a new branch, it will not take into account the extension of existing vessels, which is the main component in established tumours.

Reinhold: We had a hard time finding capillary sprouts in tumours. I support Julie Denekamp’s point that vessels existing in the tumour or pre-existing in the tumour periphery are likely to adapt to form a major part of the essential vasculature of the tumour.

Denekamp: If the tumour vessels branched better, tumours wouldn’t have so many nutritional problems. It is because the three-dimensional array is poor that tumour cells are hypoxic and resistant to chemotherapy and radiotherapy.

Reinhold: All experimental tumours investigated so far are like that. Prescott: We still haven’t addressed the point of whether endothelial prolif-

eration must automatically follow migration. When considering this we must also keep in mind that endothelial cells exist as a contact-inhibited monolayer. If a comparison can be made between capillary and large vessel endothelium, there are studies which are relevant. When a wound is small enough it can be covered by endothelial migration alone. Reidy & Schwartz (1981) found that after a very small circular injury not only was the wound covered by migration alone, but there was no proliferation of endothelial cells behind the wound, implying that cell-to-cell communication was not altered in such a way that proliferation was initiated. When a wound is larger, not only does the lead migrating endothelial cell eventually proliferate, but the cells behind it also proliferate. Thus we are dealing with two phenomena which initiate proliferation-extensive endothelial spreading and disruption of normal cell-to-cell contact.

Le Douarin: In the corneal assay why can’t one distinguish between the chronology of these two mechanisms, migration and proliferation? Why is it

ENDOTHELIAL CELL MARKERS 181

not possible to see whether the cells of the endothelial wall which produce growth migrate before they proliferate?

Folkman: Dianna Ausprunk made sequential electron microscopic auto- radiographs of neovascularizing corneas and showed that migration preceded proliferation, temporally and spatially (Ausprunk & Folkman 1977).

Ross: George Odland and I studied epidermal regeneration in human skin. We made circular rotating punch-biopsy wounds in medical students’ skin. The basal cells re-oriented from perpendicular to horizontal. They phagocytosed the fibrin clot as they migrated across and the lead cells never proliferated. It is the cells further back that make DNA and divide.

Folkman: If you put those cells into culture and they were moving around, they could still divide, couldn’t they?

Ross: That is right, but there is an analogy. The endothelial cells of the capillary loop are contact-inhibited, just like the basal cells of the epidermis. The basal cells remain attached; they never detach from their neighbours but migrate as a sheet, just as the endothelium does. It would seem that the forces are similar in the epidermal basal cells and the endothelial cells.

Denekamp: But I don’t think that in skin the leading cells never divide. The mitotic wave starts 1 mm away from a cut edge but it moves inwards.

Endothelial cell markers

Auerbach: A central hypothesis in our laboratory has been that even among capillary endothelial cells there is variation, depending on where they are and in what order they develop. Nicole Le Douarin suggested in her monoclonal antibody studies (see p 91) that very early in development there is a stem cell population which may move all over the place. Whether they are initially migratory or not, it seems reasonably well established that capillaries develop within the mesenchymal rudiments of the embryo as the rudiment develops. If there are different rudiments making different organs, is it not reasonable that there will be differences between the capillary endothelial cells that reflect those organ differences?

Besides making some general markers for endothelial cells (e.g. anti- angiotensin-converting enzyme), we have begun to investigate that question by making immunological reagents that are organ-specific. For example, we have made rat ovary cell suspensions, injected them into a mouse, fused the mouse spleen with NS1 myeloma cells and screened for monoclonal antibodies that recognize ovary. We screened on bovine ovary endothelium and subsequently tested a panel of 10 different cells, including the Chinese hamster ovary fibroblast. We have now selected five variants which bind Chinese hamster ovary fibroblasts as well as mouse and bovine ovary endothelium. That is,


ovary endothelium shares ovary antigen with other ovary cells. We have done similar experiments with brain and lung.

In the brain we have gone a bit further. A mouse brain endothelial cell line has been developed which expresses Thy-1, which is an antigen shared between brain and thymus. The mouse brain endothelium has Thy-1; mouse ovary endothelium and mouse epididymal fat-pad endothelium do not have it. We have also used the mouse brain endothelial cell line as an immunogen in rats and generated a rat anti-mouse brain endothelial antibody which detects antigens on mouse brain.

There are clearly, then, organ specificities in endothelial cells. It would be attractive to believe that those specificities arise quite early and have developmental significance; but it is not precluded that they arise secondarily. This now allows us to think somewhat differently about the inflammatory reactions induced by lymphocytes, about the specificity of angiogenesis, and about tumour cell organ specificity in metastasis. If there are endothelial cell differences in different organs and if lymphocytes are sensitized against specific organ antigens, then, when they reach those organs and see the antigens on endothelial cells, they will secrete lymphokines, amongst which are those that cause cell migration or cytokinesis and cell division. Similarly, if a tumour cell can recognize that a particular endothelial cell is different from another one, this may serve as a potential basis for selected metastasis.

Finally, the angiogenesis systems we have all been using have been non- specific. It is time to ask not whether our mitogen or your mitogen, or our cytokinesis factor or yours, causes an effect on endothelial cells, but what a factor isolated from a brain tumour will do to the brain endothelium compared to the kidney endothelium. What will an ovary tumour extract do? What will a lymphocyte immunized against brain do against brain as compared with non- brain endothelium? When we think about blood vessel development we ought to think about specificity differences as well as about common features among different endothelial cells.

J.A. Bevan:Just as there is obviously heterogeneity of endothelial cells, so there is a similar level of heterogeneity, if not greater, of vascular smooth muscle. One can look at a whole series of characteristics, including the capacity for developing myogenic tone, the subtypes of receptors for drugs, the ability to produce prostaglandins, the excitationxontraction coupling mechanisms, the properties of the cell membranes, and so on, and find remarkable variation in different vascular regions with vessels of different size and type. So, in talking about vascular smooth muscle, one must always think in terms of specific regions-it is virtually impossible to make any generalization.

Le Douarin: I am surprised that you found Thy-1 on endothelial cells. It is usually carried by neurons, I thought, not endothelial cells.

Auerbach: We were surprised too. We have a mouse brain endothelial cell


line which stains with converting enzyme and with other anti-endothelial cell markers. Using flow cytometry as well as fluorescence microscopy, we have been able to show that the cells label with Thy-1.2 but not Thy-1.1, so there is a clear specificity control for the Thy-1 marker.

Le Douarin: If it is a widespread marker it is no longer a marker at all. Thy-1 is found on many cell types.

Auerbach: As the methods improve, almost every immunological marker is being found in more places. But there are major quantitative differences in expression, and brain and thymus mark intensely with anti Thy-1 reagents. Thus while you can show good marking of mouse brain endothelial cells in immunofluorescence sections and by flow cytometry , other endothelial cells don’t show comparable staining with the reagents.

Le Douarin: The amino acid sequences of mouse brain Thy-1 glycoproteins have been shown to be homologous with those of variable-region immunoglobulin domains. It is suggested that Thy-1 may be like the primordial immunoglobulin domain (see Williams & Gagnon 1982).

Wolpert: Have you got different cultures which show these different antigenic properties?

Auerbach: Yes. Wolpert: And with smooth muscle when you culture the cells, do they

maintain their regional properties? J.A. Bevan:I don’t think that has been shown for most of the characteristics

that I have talked about. Gordon: Some of the phenotypic characteristics of vascular smooth muscle

cells change during culture (Chamley-Campbell & Campbell 1981, Ager et a1 1982, Martin & Gordon 1983).

Auerbach: The change depends on the passage number. Thy-1 has dis- appeared by passage 12.

Folkow: Each vascular bed has its own characteristics of endothelial permeability, besides the differences in flow conductance, combined capillary surface area, etc. Endothelial permeability is known to vary even along the same capillary tube. It is well established that permeability increases towards the venular end, and that the venular endothelium has a ‘microcontractile system’, which in some venules allows for the additional opening of so-called capillary ‘large pores’ (or capillary leaks). These are important for protein transfer, both normally and during inflammation, for example, when many more open up under stimulation from histamine, bradykinin, etc. Thus the capillary endothelium is highly differentiated, both along single capillaries and certainly between different vascular beds, where the presence of fenestrated (e.g. glands), continuous (e.g. muscles) and tight capillaries (e.g. CNS) is well known.

Young: Professor Auerbach, do your findings mean that any endothelial cell


in a blood vessel in, say, a metastasis from the breast to the brain would show the antigenic pattern of brain vascular endothelium?

Auerbach: Nobody as far as I know has looked at the antigenic phenotype of endothelial cells associated with tumours in different sites. That needs to be done.

Fraser: The morphology of endothelial cells can be changed. In metastatic tumours from organs with fenestrated endothelium to organs with non- fenestrated endothelium, the endothelium is fenestrated-that is, of the primary site type.

Auerbach: Does it take endothelium with it or does it induce it? Fraser: It is not clear. The tumour may induce it, but the endothelium is

certainly changed to that of the primary site. Wolpert: We are really talking about what I call non-equivalence and you call

differential specificity. To what extent does the connecting-up of the sympathe- tic nervous system to the vascular system show any sort of specificity?

J.A. Bevan:The innervation to the blood vessel differs in different organs- in density, in the distribution of the terminals within the vessel wall, and in the separation between nerve terminals and the smooth muscle cells. The amount of separation is more a function of vascular diameter than vascular region (Rowan & Bevan 1983). The dilatation innervation also varies. There seem to be dilator nerves in most regions of the body; probably the nature of those nerves differs in different regions.

Wolpert: In the same way as one has motor control that has quite a lot of specificity, is there local motor control, controlled centrally, of different parts of the peripheral vasculature?

J . A. Bevan:Y es. Wolpert: That requires a high degree of organization because the nerves may

have to recognize which bit of the vasculature they are dealing with. Is that accepted physiologically?

Folkow: To a great extent, though not at all in a ‘one neuron-one smooth muscle’ fashion. The adrenergic vasconstrictor neurons act on vascular smooth muscle cells rather as a commander commands troops. Thus, by a system of extensive though precisely delineated divergent innervation, a limited number of vasoconstrictor neurons can selectively convey central commands to, for example, all precapillary resistance vessels in one tissue, to allow for neurogenic adjustments of resistance here, with adjustments to a different extent in another tissue etc. However, as each individual vessel with its smooth muscle effectors is also under a complex local control and, further, exhibits its own myogenic activity, the end-result is an interesting interaction between a basic ‘home rule’ and a superimposed ‘governmental principal direction’, where the latter oversways the local fine-control system only in emergencies (cf. Folkow 1982, p. 430).


Glaser: Even within vascular beds there are apparently big differences. Heltianu et a1 (1982) recently found big differences between histamine receptors on the arterial side of the capillary bed and those on the venular side. There were many more histamine receptors on the venous side of the capillary bed.

Le Douarin: What about the formation of the blood-brain barrier? Are there tight junctions in the endothelial cells and when are they established?

Folkow: That question is very complex. In most brain capillaries, for example, the junctions between the endothelial cells are absolutely ‘tight’ and don’t allow anything to pass, except in a few places in the brain where there are fenestrated capillaries instead, which allow even polypeptides to pass. These areas represent a type of ‘window’ for the brain, whereby its function can be modulated by agents in the bloodstream, such as angiotensin. If at these sites there are neurons responding to such agents, they may convey widespread changes of brain function, even though the blood-borne agent has access to only a very restricted brain part.

Glaser: Some experiments on the eye are interesting in relation to that question. The retina has two circulations. The retinal circulation which I talked about earlier has typical tight junctions and is very similar to the central nervous system vasculature. The choroidal vasculature below the retina is a very lush vasculature that does not have tight junctions but is fenestrated. When a laser blows a hole in the basement membrane separating the retina from the lush vasculature underneath, vessels will start to grow in from the underlying choroid. When they first grow in they remain fenestrated but within a matter of days as they enter the retina they close their fenestrations and take on tight junctions. Eventually they just look like the retinal vasculature.

Young: We are now drifting away from ab initio antigenic differences in endothelial cells to suggesting that having started the same they are going to change. They are changing how tight their junctions are and what they secrete, and they are probably changing antigenically as well.

Wolpert: We are at the beginning of these sorts of questions. We know little of the clonal origin of the endothelial cells. Although Frank Manasek said he thought the endothelial cells didn’t move into the heart, for example, I don’t think he really knows where those cells come from.

Manasek: That is correct. Young: At the beginning of this discussion we were suggesting that there

were unalterably different populations of endothelial cells. Auerbach: The method of selecting antibodies that I described screens for

antibodies at the cell surface; thus we select for cell surface markers. These may well vary in development but no developmental study has been done yet on any of our markers.

Clqf: Another expression of cell surface differentiation is hormone receptors. There are a number of examples where endothelial cells and blood vessels


are acting as target organs to specific hormones. Reactivity to these hormones can differ in different sites of the body. This could well be another approach. Some of the antigens you are picking up may be related to hormone receptors.

Denekamp: I understood that Bob Auerbach felt that the capillary in certain tissues is derived from the primordial cells of those tissues and didn’t grow out from the tubes that started off the vascular tree. How does that relate to Nicole Le Douarin’s work showing that all the vascular elements have a common antigen?

Auerbach: The literature is replete with things I have written and Nicole has disproven! In almost any developmental study we begin at a certain point and describe what we see. If an organ rudiment on day 12 or day 13 does certain things, that tells us nothing about what happened 24 hours earlier. In embryogenesis everything at some point presumably came from somewhere. If there is early migration of cells into the rudiment, that would not be picked up by any study that then analyses that rudiment.

Denekamp: Are these two pieces of information compatible? Have all the capillaries been formed by outgrowth from the vascular tree, or can tubes form and then hook into the main system?

Le Douarin: A certain kind of marker is needed to follow the process from the very beginning. Without such a tool many misinterpretations may arise. I think we have to wait for more to be done on the very beginning of the development of the vascular system.

Morphogenesis

Wolpert: We should now move from differentiation and non-equivalence back to morphogenesis. The most important thing I have heard at this meeting relates to tension. Sprouting is clearly a key process in the morphogenesis of the vascular system and that is largely about the generation of tension. The cell leaves the vascular wall and drags other cells behind it. Tension is generated by cells within the capillary or within the vessel wall. It thus leads to the formation of the sprout and it may also stimulate cell multiplication.

The second aspect of tension is related to what Professor Folkow was speaking about. That is, tension is generated by pressure within the vessels and the cells somehow adjust their numbers so that each of them is under the same tensile stress.

I would say that the cells may care less about chemical factors than about tension.

Le Douarin: What is the factor which makes the cell leave the wall and induce the tension?

Manasek: The question can be reduced even more than that. You can ask

MORPHOGENESIS 187

why does a particular cell or particular group of cells decide, if you want to use that term, to initiate a sprout. That can only be approached simplistically. There are a few possible reasons for this. I would like to think that perhaps those cells are more deformable or have a higher compliance. If they are more deformable one can make certain predictions about their internal architecture, and one can get at this with present-day techniques.

With Yushi Isobe and Professor Yutaka Shimada in Japan we are looking at regional differences in the cytoskeleton in the wall of the very early developing heart. By transmission electron microscopy and light microscopy these cells seem to be all identical. We have recently obtained confirmation that there are qualitatively different cytoskeletons in different regions of the heart. This is a repeatable observation. I would like to propose that the sprouts may also have differences that alter their compliance and that minor differences in pressure within the wall or within the lumen will produce a morphological deformation. This is a self-perpetuating process.

Berry: The vessels are all anisotropic, aren’t they? The mechanical properties longitudinally are quite different to those tangentially. That seems to be true of all classes of vessel. When vessels have to store energy in big loads it is done by a load-shedding structure. Teleologically it looks as if some effort is made to maintain that.

Manasek: There certainly is anisotropy in the wall. All you need to do is to change that in one area and make it isotropic with a slightly higher compliance. That portion of the wall will then behave very differently. Perhaps a sprout is like an aneurysm.

Young: If the sprouts appear randomly, as was suggested earlier, you need only a random distribution of weak endothelial cells, don’t you?

Manasek: Yes, all you need is a random distribution of the signal or whatever it is that alters the cytoskeleton, thereby altering the ability of the wall or cell to resist deformation.

Hicks: One could easily produce a unified hypothesis (which may or may not bear any relation to fact), whereby any factor stimulating sprout formation reacts with the cell membrane to alter the physical arrangement of its lipid phase. This will alter its fluidity and allow a mechanical bulge in that membrane. For example, if the state of the lipid is changed from a smectic mesoph- ase to a micellar arrangement there will be a completely different tension in the membrane. Changes of this sort are known to have far-reaching implications in terms of activating membrane enzyme systems. Like the effect of promoters on carcinogen-initiated cell membranes, there could be a knock-on effect, or a cascade of reactions which in turn could control a genetic switch within the cell.

Glaser: Microaneurysms in the microvasculature of the retina occur com- monly in diabetics, in patients with hypertensive retinopathy, and in several other disorders. From some of the older studies it was suggested that in the


diabetics these microaneurysms are abortive attempts at neovascularization. It has not been proved one way or another but the microaneurysms are all over the eye and only in very discrete locations do you see neovascularization. Patients with hypertensive retinopathy have just as many if not more of these microaneurysms and we never see neovascularization. There is thinning of the wall and the microvasculature bulges, but there are no sprouts.

Wolpert: From Judah Folkman’s description of the action of heparin in making cells move more, my guess is that it is going to be a more subtle phenomenon. It is too important to the development of the vascular system to be just mechanical.

Ryan: But mechanical factors are important in chemistry! Any molecule which looks like a tight spiral will pull apart when it is stretched. It is still the same molecule but its stereochemical shape, and its capacity to pick up loose inhibitors, activators or agents which will cleave it in two, alter with the change in shape. The moulding of fibrillar material depends on such factors as well as on attachment or binding at either end. We have to consider molecular configuration too.

Fraser: The endometrial vasculature grows very rapidly and it re-grows every month. That must surely sprout from muscular-type arterioles, as most of the capillary bed is lost.

Folkman: I think that Arthur Hertig in his classic 1935 monograph on angiogenesis in the placenta showed that arterioles originate from capillary- like structures. I don’t know of a study of the menstrual cycle. Cliff: Schoefl(l963) showed that the first event in the formation of the sprout

in the cornea is the dissolution of the vascular basement membrane. I also wrote a paper (Cliff 1963) on vascular sprouting. The first event in the matura- tion of the sprout towards becoming a recognizable vessel is the development of the basement membrane. This is a cardinal event in the expression of the differentiation of the vessel and in the maintenance of that vessel as an independent structure.

Kuettner: Do all capillaries have basement membrane? Folkman: New or growing capillaries often don’t, but mature capillaries do. Kuettner: Where is formation of the new vessel happening? Folkman: Usually at the tip of a sprout or at the apex of a loop. Cliff: In inflammation when white blood cells leave the endothelium they

often get trapped beneath the basement membrane. It takes quite a lot of effort for them to get out. There is a strong mechanical barrier.

Wolpert: There are many things we could still discuss, for example Dr Fraser’s comment that the development of the vasculature in pathological situations such as tumour formation is quite different from the embryonic development of the vasculature. The whole relationship between the embryonic development of the vasculature and other situations remains totally

MORPHOGENESIS 189

obscure and I am not sure that we have a great deal to say about it at this stage. There is also the question of how sprouts find each other. So it is appropriate that we should end this discussion with something from Judah Folkman.

Folkman: I would like us also to think about the other half of what the embryo is doing in vascular development. Angiogenesis is the building up of the vascular system, but vascular regression is also going on. If one implants into the rabbit cornea a polymer pellet containing tumour extract, new capillaries grow into the cornea. If the pellet is then removed, the new vessels regress and eventually, in one to two months, disappear. However, the earliest event in the regression seems to be adherence of platelets to endothelium near the tip of a capillary. Platelet sticking occurs within the first day after removal of the stimulus. There is stasis and then flow stops over the next few days. Capillary endothelial cells die, and macrophages arrive and digest them. Is it possible that one of the functions of platelet factor IV is to initiate regression of new capillaries?

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General discussion I11

Endothelial cell regeneration in hypertension and hypercholesterolaemia

Prescott: Many methods have been reported for wounding the endothelium of large vessels, including balloon catheterization, freeze-drying and infusion of hypotonic solutions. Such procedures have produced not only extensive areas of endothelial denudation but also damage to the medial smooth muscle cells. Recently Reidy & Schwartz (1981) developed a model which produces a small superficial intimal wound only a few endothelial cells wide. In this model a filament enclosed in a protective sheath of tubing is inserted into the femoral artery of the rat and advanced to the level of the diaphragm. The filament is then pushed out of the tubing, producing a longitudinal injury. We used this method to produce a wound about six endothelial cells wide (Fig. la) . Trans- mission electron microscopy five minutes after wounding shows platelets adhering to the denuded area. The internal elastic lamina remains visually intact and there is no obvious morphological damage to the medial smooth muscle cells.

I was interested in using this technique to study endothelial cell regeneration in hypertension and hypercholesterolaemia, since these two states are known to be risk factors for the development of atherosclerosis. Morpholo- gical alterations in endothelial cells have been reported in both conditions. In addition, hypercholesterolaemic serum has been shown to inhibit endothelial cell migration in culture. It therefore seemed reasonable to suggest that these two risk factor conditions might interfere with the repair of endothelial cell injury.

Four groups of rats were wounded at 22 weeks of age. The control group was a Sprague-Dawley-derived strain. The second group consisted of control animals made chronically hypertensive by the single clip Goldblatt technique.

The third was the RICO strain of genetic hypercholesterolaemic rats (Mul- ler et a1 1979) and the fourth consisted of RICO genetic hypercholesterolaemic rats made chronically hypertensive by the Goldblatt technique.

To our suprise, in all four groups endothelial repair not only followed the same sequence of events, but also there were no significant differences in the time it took for complete healing to occur. In all groups the first event was endothelial cells migrating into the wound (Fig. lb) . These cells were not labelled by autoradiography and had increased surface areas. By 24 hours

191

FIG. 1. Scanning electron micrograph of wounded thoracic aortae. Endothelial borders stained with silver. (a) 5 min after injury. (b) 18 h after injury, endothelial cells migrating into wound.

ENDOTHELIAL CELL REGENERATION I N HYPERTENSION 193

endothelial cell continuity was restored but the endothelial cells were randomly oriented along the former wound line, as opposed to neighbouring non-wounded areas where endothelial cells were seen to be lined up in the direction of blood flow. By autoradiography it could be seen that thymidine labelling rarely occurred in the area of wound closure but rather was mainly in the area of the original wound borders. Twelve hours later the endothelial cells were longitudinally arranged in a formation reminiscent of a zipper (Fig. 2a). At this time most of the endothelial cells along the wound line were incorporating thymidine. Subsequent endothelial proliferation resulted in a line of narrow densely packed endothelial cells along the former wound (Fig. 2b). Sixteen weeks after injury a line of narrow endothelial cells remained. However, at this time the cell density was much less than that seen shortly after wounding.

We used scanning electron microscopy to see the former wound line and then made transmission electron micrographs of that area. In all groups, endothelial cell continuity was restored without the development of any intimal thickening (Fig. 3).

These results are consistent with both in vivo and in vitro wounding studies showing that if any injury is small enough the wound is covered by endothelial cell migration alone (Sholley et al 1977, Reidy & Schwartz 1981). If the wound is larger, endothelial cell proliferation is necessary for closure. Our wound was closed by a combination of proliferation and migration. We found that endothelial cell proliferation extended only a few cells back from the original wound borders. After balloon-induced injury, however, Schwartz et a1 (1978) reported increased endothelial cell proliferation far removed from the wound borders.

This tempts me to speculate that not only is the signal for endothelial cell proliferation dependent on the size of the injury but also it may be transmit- ted differently in the longitudinal and circumferential directions. Relevant to this is recent work by C. Haudenschild and S. Harris-Hooker (personal communication) on cultured endothelial cells in which injury to a single endothelial cell produced changes in endothelial cell motility 20 cells dis- tant-rather like a pebble thrown in a lake having a ripple effect.

In summary, our findings indicate that, in the rat, hypertension and genetic hypercholesterolaemia do not interfere with the endothelial cell repair response after a narrow superficial denuding injury.

Roach: Was the injury made on the dorsal or ventral surface of the aorta? Prescott: Unfortunately, what goes up must come down. We pushed the

filament up the dorsal surface and found that we could not pull it back along exactly the same wound line. Thus we had to rotate the filament so that the wound line formed coming down would be as far away as possible from the original line. We found by experience that after rotation the pulling-down line

FIG. 2. Wounded thoracic aortae. (a) 36 h after injury; wound closed with narrow endothelial cells aligned longitudinally. (b) 7 2 h after injury; dense line of narrow endothelial cells covering previous wound.


FIG. 3. Transmission electron micrograph of the previous wound in a RICO rat 15 days after injury.

was on the ventral surface and that this line was straighter and narrower. Maybe our hands were steadier coming down. It was this narrow ventral line which was analysed.

Roach: You didn’t compare one with the other? Prescott: No, because quite frankly the dorsal line was not totally straight

and did not have the nice clear margins which I showed you. Roach: It is interesting to speculate whether the difference was because one

wound line was stretched more than the other and whether that played a role in how the cells migrated and covered the lumen.

Prescott: We were interested in assessing that but the wound produced on the dorsal surface was not consistent enough to be analysed. Cliff: I am surprised you didn’t get more involvement of blood platelets,

leucocytes and things like that. It seems incredible that you can strip away an area of aorta which in the rat is really quite significant. Is there no involvement of the thrombotic system or any signs of inflammation?

Prescott: Immediately after injury there is an incomplete monolayer of platelets sticking to the wounded area but not a complete carpet. We found no white cell interaction at all.

Ross: One cannot induce atherosclerosis in the rat without taking very special precautions because of the differences in protein profiles and in lipid metabolism and other aspects that are not well understood. That doesn‘t deny the beauty of the experiments for understanding how endothelial regeneration occurs, but they have to be applied to angiogenesis with caution.


Prescott: Absolutely, but it has been shown that intimal thickening consist- ing of increased amounts of extracellular material, blood-derived cells and smooth muscle cells occurs in rats with hypertension and hypercholesterolaemia. Such thickening, though, is not an atherosclerotic plaque.

Bur: In your pictures the endothelial cells which migrate first are very large. The cells which appear next are smaller. Are the small cells a new generation or have the existing large endothelial cells changed their form and size?

Prescott: The cells which at first migrate eventually proliferate and the final line of very narrow densely packed endothelial cells comes after this proliferative event.

Reinhold: Would this be a suitable system to test for anti-angiogenic factors?

Prescott: The system is in the aorta so again there is the problem of capillaries versus large vessels. Corneal vascularization or in vitro migration assays are much more appropriate techniques.

Glaser: What happened to the platelets when the endothelial injury was covered over?

Prescott: In one out of 18 rats analysed 24 hours after injury I saw platelets within the intima after endothelial cell closure. I was rather surprised because I remembered that Malczak & Buck (1977) stated that advancing endothelial cells clear away platelets as they cover an injury.

Berry: We have looked at about 14 rabbits which one of our cardiovascular surgeons is using to develop new stapling techniques for coronary artery by-passes, again veins to arteries. Very seldom does one find platelets packed under the endothelium. The system seems to be similar to the epidermis in wound healing. The epidermis makes a level at the secure bed of granulation tissue deep to the debris. I imagine it has to grow on the proper substrate, as it were, so a cell stuck on non-viable material may ‘invade’ instead.

Denekamp: How far away did you observe proliferation? Prescott: We found increased proliferation only a couple of rows back from

the original wound margins. This is in contrast to Schwartz et a1 (1978) who found increased proliferation as far away as 100 cells downstream from a wound produced by a balloon.

Denekamp: Were the cells only downstream? Prescott: The study by Schwartz et a1 only analysed endothelial cell regen-

eration in adjoining intercostals and from the aortic segment below the injured area.

Folkman: Professor Wolpert, there must be other systems in which the front layer of migrating sheets or migrating cells in a wound do not divide. J.P. Trinkaus at Yale has looked at this in amphibian wounds. At the level of the cell, why do you think the front row of cells can’t undergo division whereas in sparse cultures cells are migrating and dividing all the time?


Wolpert: I have nothing useful to say about that. I was thinking about it in relation to your remarks about a single file of cells. Maybe it has something to do with the end cell being free and so able to undergo locomotory activity by putting out processes. This may somehow block proliferation.

Hicks: The leading cell has a free edge. Does the lack of contact inhibition at one face have something to do with differentiation versus division? Does it prevent the leading cell from dividing?

Wolpert: The whole idea just doesn’t hold. It doesn’t fit with the idea that when these cells are put into sparse cultures and move around they are perfectly capable of dividing.

Folkman: These cells are all in the same plane. Wolpert: It is a mystery that we just don’t understand. Ross: Judah Folkman was referring to attached cells in a sheet. Non-

attached cells move around, but before they enter mitosis they stop moving and round up. In the sheet the cells are advancing and perhaps they cannot assume the proper configuration.

Denekamp: Margaret Prescott is looking not at mitotic cells, but at cells taking up tritiated thymidine-i.e., which are 8-10 hours pre-mitotic. Those cells don’t round up in culture.

Wolpert: The rounding up is a later phenomenon. It occurs at mitosis. Ross: That’s right, but what she is looking at has to precede the rounding. Folkman: Is it a fairly general phenomenon? Wolpert: The only examples I know are the ones related to endothelial

cells. Does this happen when you wound 3T3 cells in culture? Ross: If 3T3 cells in a confluent culture are wounded, the individual cells

march freely out into the wound and do not attach to the other cells, whereas endothelial cells never part from one another but migrate out as a sheet.

Denekamp: How common is the hyperplasia in vessels after different sorts of trauma? Margaret Prescott has shown that after surgical injury densely packed endothelial cells persist for 16 weeks. When we injure the endothelium by irradiation, however, we see reduced endothelial cell numbers, persisting up to 12 months (Hirst et a1 1980).

Prescott: Increased endothelial cell density after wounding large vessels in rats has been reported, but to my knowledge the longest time point studied was 26 weeks (Hirsch et al 1981). Possibly such hyperplasia is species- specific, as similar wounding in rabbits does not result in such a response (Ramsay et a1 1982). I could find no documentation of endothelial ‘scars’ in humans.

Ryan: Adjacent to the basal lamina of epidermis, mitosis may occasionally occur at rest, but there is a stage during proliferation at which all the mitotic figures get higher and higher up in the epidermis through jostling in the basal layer. The epithelial cells can’t really be migrating inwards because there is a


barrier preventing them doing so. Jostling and the final disposition of cells is more complicated than can be explained simply by migration in the front line (Ryan 1975).

A hypothesis of atherogenesis

Ross: In our laboratory we have established a hypothesis of atherogenesis called the response to injury hypothesis, which is a modern version of many of the things that Rudolph Virchow suggested in 1865. We are doing a series of correlated in vivolin vitro studies in the pig-tailed monkey, Macaca nemesf- rina, and in humans. The non-human primates are fed on a high fatihigh cholesterol diet with eggs and butter added to the diet. The plasma cholesterol levels go up to 50&800rng/dl, which is a criticizable level, but I would have to live to the age of Methuselah if I kept them on the usual level of 250-280 mg/dl, which is a hypercholesterolaemic level in humans. The whole process is accelerated by this diet, but I don’t know whether it causes other changes.

We did both scanning and transmission electron microscope studies on these animals, starting two days after they began the diet. We then looked at the animals in each group at one week, two weeks, one month and at monthly intervals for up to two years. Each animal is given tritiated thymidine at 19, 7 and 1 hour before being killed. We are examining endothelial turnover in the animals and platelet survival levels are studied monthly; many other studies are also being done. We also did cell culture studies on the platelet-derived growth factor that we discovered in 1973 (Ross et a1 1974). Some of the cellular and molecular approaches were taken to try to understand how that factor works biologically at the cellular level and what role it plays in the genesis of the lesions of atherosclerosis.

In a normal monkey the endothelial cells at a bifurcation in the aorta are flat and form a continuous monolayer. They take the direction of the flow of blood. After the animals have been on the diet for three to four weeks one begins to see the equivalent of the fatty streak seen in humans. In the transmission electron microscope we can see subendothelial macrophages or foam cells containing lipids lying under a continuous endothelial cover. Each of these was originally a monocyte. Occasionally we see some smooth muscle cells with lipid. After a longer time we begin to see changes in the fatty streaks. At the bifurcations we begin to see the lobular appearance they create at the luminal surface of the aorta at a very low magnification in the scanning electron microscope. There is some intrusion on the possible flow of blood by excrescences in the artery wall. After four months we can see the lobulation that occurs under an intact endothelium due to the presence of the

A HYPOTHESIS OF ATHEROGENESIS 199

lipid-laden macrophages, and there are monocytes that attach in numerous places on the surface. There are a few neutrophils around as well.

At four to five months we also begin to see areas where the subendothelial macrophages are re-exposed. In some cases these macrophages appear to be extruded into the bloodstream. In fact, starting at about three months, we see increasing numbers of lipid-laden macrophages in buffy coat smears. These increase in number as time goes on. We suggest that in many cases this represents an egress of the monocytes that originally entered the artery wall, took up lipid and came back into the bloodstream as effete lipid-laden cells. This is similar to what R.G. Gerrity reported about a year ago.

At between five and six months we begin to see what we had originally expected to see earlier. That is, there is endothelial desquamation and denudation, which first appears at the iliac bifurcation and in the leg arteries. By seven to nine months we see it in the abdominal and lower part of the thoracic aorta. By 10-12 months we see it in the arch of the aorta and after 12 months we see it in the coronary arteries. These changes seem to work their way up the arterial tree. The animals, incidentally, are all fixed by perfusion at normal pressures. What we see is a fatty streak under endothelium and an area of clear endothelial desquamation and denudation, with platelets. We also see attached monocytes and in some cases attached foam cells. The entire area of exposed subendothelium is covered with a mural thrombus of platelets. The only way one can explain such platelet adherence in a perfusion-fixed animal is that the platelets were there before fixation. At somewhat later intervals we again see macrophages and platelets. After about five months these areas develop, though somewhat slowly, into fibrous plaques that by one year are so similar to the lesions in humans that we can scarcely tell them apart.

So there is the same kind of evolution in the monkey as we see in humans. There is a fatty streak early on with little or no smooth muscle proliferation, though there may be a couple of layers of smooth muscle cells under the macrophages, under an intact endothelium. Eventually there is an extensively advanced lesion.

In our injury hypothesis we suggested that some form of ‘injury’ to the endothelium-be it mechanical, chemical, immunological or what have you- leads to endothelial cell-connective tissue detachment and, at particular anatomical sites such as branching sites and bifurcations, perhaps to desquamation and denudation, which exposes the subendothelial connective tissue to macrophages and platelets. We suggested that this leads to migration of smooth muscle cells through the fenestrae of the elastic lamina into the intima, and to proliferation. We also suggested that if the injury ceases the lesions can regress. If the injury continues over 10, 20 or 30 years one gets a slowly progressive lesion.


What we see in the hypercholesterolaemic monkey is attachment of monocytes at such sites and subendothelial localization of these monocytes with a bit of smooth muscle emigration. Then some of these monocytes begin to break through the endothelium and re-enter the bloodstream. By five months we see monocytes, macrophage localization, platelets and then massive migration and proliferation of smooth muscle cells, with progression into the classical lesion that I have just described.

What role might platelets play in this process? In 1973 we discovered that platelets contain an extraordinarily potent mitogen, the platelet-derived growth factor (PDGF) (Ross et a1 1974). Some eight years later we purified PDGF to homogeneity. We now purify about 300,ug a week. The process takes about five weeks, starting from out-dated human platelets in mixed plasma. We can radio-iodinate this purified material and we have measured its binding characteristics to a line of 3T3 cells with about 300 000 receptors and to normal 3T3 cells with about 130000 receptors, monkey arteriolar smooth muscle cells with 80 000 receptors, human foreskin fibroblasts with SO 000, human arterial smooth muscle cells with 40 000 and a line of 3T3 cells that we have mutated and that have about 1000 receptors. They all bind in a saturable fashion with an apparent dissociation constant of 10-l' M.

This factor therefore binds with very high affinity to a specific cell surface receptor. We also looked at the correlation with increased thymidine incorporation. Binding of the ligand to its receptor leads to DNA synthesis and cell multiplication.

PDGF is a very cationic protein; it is hydrophobic, with a relative molecular mass (M,) of about 30000. It consists of two chains that are disulphide- bonded, the disulphide bonds being necessary for biological activity. It does many things when it binds to cells. In seconds it induces phosphokinase activation of tyrosine-containing proteins. Specifically the receptor for PDGF may be phosphorylated on tyrosine. In addition a whole host of cytoplasmic proteins are phosphorylated on tyrosines, threonines and serines. It also induces rapid sodium-potassium ion fluxes. Within less than an hour it induces increased phospholipid turnover, increased endocytosis, increased binding of low density lipoprotein (which is the principal cholesterol-carrying moiety in the plasma) and increased protein and RNA synthesis. As Grotendorst et a1 (1981) have shown, PDGF is chemotactic. It is the only growth factor that has been isolated and purified, so far, that is chemotactic for smooth muscle cells. This potentially explains why smooth muscle cells are attracted from the media into the intima after platelets bind to the surface.

The question remains of what role PDGF potentially plays in humans. V. Gregory Brown and his colleagues at UCLA have studied nearly 200 patients who have had coronary by-pass surgery (unpublished). It is now well known that 3.5% of all by-passes (though not 35% of all patients) re-occlude within a


year. Re-occlusion is due to the formation of a new lesion of atherosclerosis at the peri-anastomotic site where the saphenous vein is tied into the coronary artery. Brown and colleagues decided to use the opportunity to do a clinical trial. At the University of Washington a computer-based program has been developed to take two views of coronary serial angiograms so that the amount of occlusion due to new lesion formation can be quantified. The computer program straightens the two views out and can calculate the amount of interference or occlusion. By taking such serial angiograms from patient to patient one can get good quantitative correlative data. Brown et a1 did a double-blind study in which they put the patients on either a placebo, aspirin- dipyridamole, which is a very potent anti-platelet regimen, or aspirin alone for a year. This is the first clinical trial to ask the specific question: what will anti-platelet agents do to the formation of a new lesion of atherosclerosis? All other clinical trials so far have looked at patients who have had one myocardial infarct and therefore have advanced atherosclerosis and calcified lesions, which is a very different question. This study is asking what is the effect on the formation in a virgin vessel, as it were, of a new lesion. (We know the saphenous vein doesn’t get atherosclerosis normally.)

When Brown et a1 decoded their results they found that in the placebo group within a year 7% showed marked stenosis and 26% moderate stenosis. With clinical anti-platelet therapy the 7% figure dropped to 1%, which is highly statistically significant, and the 26% fell to 12%. Surprisingly, on low-dose aspirin alone the 7% dropped to 4% and the 26% to 15%. To my knowledge this is the first example that one can in fact use anti-platelet therapy in humans to prevent the formation of a new lesion of atherosclerosis, albeit in this very special place.

This raises more questions than it provides answers. From the injury hypothesis we think that the various risk factors-hypercholesterolaemia, hypertension, diabetes, cigarette smoking, age, sex and so on-all impinge in one way or another on the maintenance of homeostasis in the arterial tree. There is some form of endothelial injury, ranging from functional injury on the one hand, with simple alterations in the non-thrombogenic character of the surface of the cells and in the ability of the cells to transmit materials into the artery walls, to cell-cell detachment, cell-connective tissue detachment and desquamation on the other hand. We think this injury leads to a sequence of events of which hypercholesterolaemia may be somewhat representative. However, it is entirely conceivable that the sequence and the chronology of the sequence is somewhat altered from hypercholesterolaemia to cigarette smoking to hypertension. One would expect variations on this theme and variations in the subtlety of the alterations that lead to the development of atherosclerosis. We suspect that in some circumstances platelets and PDGF, which is an extraordinarily potent mitogen, may play a role in inducing this


proliferative lesion. In addition we know that macrophages enter the artery wall, derived from monocytes. There are at least four possible characters in this play.

The platelets are the source of PDGF, which is the only growth factor that has been purified to homogeneity that we can study. We now have antibodies to it, we have a radio-receptor assay for it, and we are in the process of being able to measure it in the plasma and urine of individuals undergoing surgery who are at risk in certain ways. We should soon begin to get some idea of the relative importance or unimportance of PDGF in atherogenesis.

In 1976 we discovered that when macrophages are appropriately activated they too release a growth factor which is as potent as the growth factor derived from platelets. It differs from PDGF because antisera to PDGF have no effect on the macrophage-derived factor. MDGF is still in crude form in our laboratory and it does not compete for binding to the receptor for PDGF. We have established an excellent source of MDGF and hope to be able to do the same kind of studies with it as we are doing with PDGF.

With Steve Schwartz a couple of years ago we also discovered that endothelial cells make a growth factor which has a number of biological and biochemical similarities to PDGF. We don’t yet know whether this is an in vitro artifact or whether it exists in vivo. This remains an even greater mystery but it is being worked on in several laboratories.

Finally, Bob Wissler’s group several years ago showed that hyperlipaemic low density lipoprotein in some circumstances may also be mitogenic. We don’t really know where this fits into the picture.

All these possible mitogens, which do much more than induce mitogenesis, potentially may play a role in inducing smooth muscle cell proliferation. I would remind you that this is the accepted key event if the lesions of atherosclerosis are to become clinical sequelae of some significance. If one doesn’t have a proliferative lesion one probably does not have to worry about occlusion and impedance of the vascular supply. If one could prevent this proliferative lesion, theoretically one might be able to prevent the disease. Using this hypothesis, which we keep modifying as we get more factual information, and using these approaches at the cellular and molecular level, we hope to begin to understand how atherogenesis comes about. Atherosclerosis is the cause of more than 50% of deaths in the UK and in the USA, so gaining some information at the cellular, molecular and clinical levels on how it takes place can be nothing but helpful.

Prescorr: Your studies further show the probable multiple causes of atherosclerosis. Endothelial cell denudation followed by platelet adherence is not a prerequisite for its development. Macrophages as well as possible functional changes in endothelial cells are also involved. Although this is what you implied, it is still very common for people to say ‘Yes, we know all about


atherosclerosis. Ross proved that the principal cause is endothelial denudation following injury’.

Ross: We are still trying to define what we mean by injury. Injury represents a whole spectrum of events. In some cases the endothelium may be completely intact but it still may be injured functionally. This may be very important. In other cases it may actually be denuded, as we now feel confi- dent it is after a particular length of time in the hypercholesterolaemic monkey. Obviously we shall never know in humans, unless we can develop markers telling us that when a certain substance appears in the plasma, a particular amount and kind of injury will occur in the endothelium. I don’t think that will ever happen.

Berry: Did you suggest that PDGF increases the synthesis of prostaglandin by smooth muscle?

Ross: PDGF specifically increases prostacyclin formation by smooth muscle cells. This might be a feedback inhibitory mechanism, although that is pure speculation. Cliff: One of the biggest problems with atherosclerosis is that the disease

never really develops in animals. In human beings it is a real and important disease with well-recognized clinical sequelae. In another American study of monkeys on high cholesterol diets, one very famous female monkey reached a cholesterol level of thousands of milligrams. She was said to have had a myocardial infarct, but the paper that was going to show the classical histolo- gy of the coronary arteries and the infarct never appeared. Have your monkeys shown any signs of circulatory impairment? Or have you got another of these cases where you are just clogging the arteries up with fat?

Ross: We shall be able to answer when we examine the monkeys, but we are not using females. We have tried to eliminate the problem of female sex hormones.

There is a stress factor also. We have monkeys in two groups in the study, a free-ranging group and a caged group. The caged group on a lower cholesterol intake have higher plasma cholesterol levels than the free-ranging group. The stress of being in a cage clearly makes a difference. It will be interesting to see whether over a long period this plays a role in the whole process.

Roach: We mustn’t forget that the lesions of atherosclerosis are patchy. We have shown (Roach & Smith 1983) that the patches occur in the same places in the cholesterol-fed rabbit, regardless of how much cholesterol we give them or how fast, as they occur in hypertension. If we modify the flow we can modify where the lesions develop. There obviously is something that relates flow to where changes occur in the wall.

Ross: I completely agree. One can speculate that eddy currents or shear forces on the endothelial cells have an effect, I think hypercholesterolaemia is not producing a toxic effect on the endothelial cells, but having a subtle but


interesting effect on the plasma membrane. Hypercholesterolaemia occurs mainly by hyper-LDL, if you will, and low density lipoprotein contains large amounts of cholesterol. Cholesterol exchanges immediately and rapidly; it requires no enzymic procedure. If low density lipoprotein comes near to a cell membrane the exchange between the lipoprotein particle and the plasma membrane is immediate. If an animal is bathed in high concentrations of LDL for a period of years, as ours are, it is reasonable to assume that the plasma membranes will contain more cholesterol moieties and more phospholipid molecules per unit area, and that the nature of the phospholipids will change. It is well known in cells other than endothelial cells that this increase in cholesterol moieties in the plasma membrane leads to increased viscosity of the membrane. To put it another way, the membranes become more rigid and less malleable.

What I have said so far is reasonably factual, though not studied in endothelial cells. But now comes the wild speculation. My feeling is that at branching sites and bifurcations and other sites that may be rheologically at risk it may simply be that the plasma membranes are no longer as malleable in response to the flow of blood and cells that is passing over them all the time, and that the stresses on the cells may be so great that if the membranes become rigid enough they may get pulled away from the connective tissue to which they are attached at the junctional sites. Of course there is constant change in the shape of the artery wall because the macrophages get under there, making the surface look like cobblestones. This must change the flow characteristics as well, particularly at branch points. This sets up a beautiful focus, because as soon as platelets see collagen or altered cell surfaces they will adhere and undergo shape changes. Monocytes and macrophages will do the same. The change in the plasma membranes of the endothelial cells in hypercholesterolaemia may be a subtle change. I don’t know any evidence to suggest that hypercholesterolaemia is toxic to endothelium and I certainly wouldn’t want to suggest that it is.

Young: Your pictures of macrophages under the endothelium producing little hummocks that might allow trauma to the endothelium are very appeal- ing. But the places where there is stretching of the artery, particularly at the knee and the groin, are the places least likely to be affected by atherosclerosis (Browse et a1 1979). The stretching must affect the endothelial profile and change flow characteristics.

Ross: I don’t think the arteries are stretched in this case. I think the lumen is intruded upon.

Young: But the parts of the arterial tree that stretch during movement develop endothelial corrugations that intrude on the lumen, yet they are the very parts that tend not to be affected by atherosclerosis.

Ross: Most of the incursions of macrophages occur at branching sites, not


in straight segments of the wall where stretching could occur. Stretching would be very difficult at a bifurcation of a branch.

Young: But the commonest site of atherosclerosis is the superficial femoral artery at the adductor hiatus in the thigh, where there are none of these handy bifurcations.

Ross: That is the commonest site of atherosclerosis that occludes, but the commonest site of lesions in these monkeys is at branching points and bifurcations.

Young: It still has to be explained. Folkman: Is there any correlation between the number of foam cells in the

buffy coat and any phase of the disease? Ross: In the monkeys we begin to see foam cells at three to four months.

The numbers increase over the next six to seven months and then seem to stay at a reasonably high level. They don’t disappear while the animals are on a high fat diet. We have not looked at what happens if we take the animal off the diet.

Folkman: And what about humans? Ross: These observations are quite recent. One needs to go back and look

at humans.

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Muller KR, Li JR, Dinh DM, Subbiah MTR 1979 The characteristics and metabolism of a genetically hypercholesterolemic strain of rats (RICO). Biochim Biophys Acta 574:334-343

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[Novartis Foundation Symposia] Ciba Foundation Symposium 100 - Development of the Vascular System...

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