Studies on retinal capillary cells in tissue culture

Vision Rewwh Vol. 21. pp. 165 to 168 Pergamon Press Ltd 1981. Printed in Great Britain

STUDIES

ON RETINAL CAPILLARY IN TISSUE CULTURE ROBERT

N.

CELLS

FRANK

Kresge Eye Institute, Wayne State University School of Medicine, Detroit, MI 48201, U.S.A. Abstract-Under appropriate conditions, it is possible to culture individual cell types from the retinal capillaries of several species. When capillaries from adult animals are isolated by a homogenizationfiltration technique, and then placed in culture medium, a monolayer of cells proliferate that are identifiable as deriving from the intramural pericytes. Thus far, identification is based largely on radioautography of the original vessel explants. When capillaries from the retinas of fetal or very young animals with immature retinal vascular systems are placed in culture, a slowly growing monolayer of endothelial cells results. identification here is based on the light microscopic morphology of the ceils together with the finding zonulae occludentes by electron microscopy, and, in preparations from appropriate species, by factor VIII immunofluores~nce and angiotensin converting enzyme activity. In preliminary investigations to date, these vascular cell cultures have been used in the studies of the aldose reductase system and of collagen synthesis, both of which are biochemical mechanisms that may be irrelevant to the pathogenesis of diabetic retinopathy. The availability of cultures of individual types of retinal capillary cells should be an extremely useful method for further studies of the biochemistry, physiology, and growth potential of the smallest blood vessels of the retina.

Abnormalities of the retinal blood vessels are central to many important ophthalmic diseases. A better understanding of these diseases, perhaps leading to improved methodS of thefapy and even prevention, would result from knowledge of the biochemistry, physiology, and growth properties of the cells that comprise the retina1 vascular system. In the past, methods for isolating the retina1 vascular network, such as the trypsin digest technique (Kuwabara and Cogan, 1960), have led to preparations that were useful for light microscopic study, but not for biochemical or tissue culture investigations. Several years ago, a method was reported for isolating in rather “clean” preparations viable fragments of retina1 (Meezan et al., 1974) and cerebral (Brendel et al., 1974) capillaries. This seemed to us to lend itself well to tissue culture, biochemical, and certain specialized anatomical studies, such as scanning electron microscopy, that could not have been performed by any other methods of which I am aware. Our work to date has yielded interesting results, with the promise of more to come. In our original study using this method, Dr. Sheldon Buzney and I found that, when retinal capillary fragments from adult cattle and rhesus monkey eyes were placed in culture, cells slowly proliferated from the explants. The cells were irregularly polygonal, and they grew in a loose monolayer without any polarity, and with some overlapping of the individual elements. Electron microscopy revealed a multitude of intracellular organelles, specifically including abundant myofilaments and micropinocytotic vesicles, but no structures that enabled specific identification of the type of cells that were proliferating. Autoradiography revealed, however, that the proliferating elements were intramural pericytes (“mural cells”) of the retinal capillaries (Buzney et al., 1975). We were struck by the fact that we never observed endothelial cell proliferation from these preparations, in particular since others (Jaffe et al., 1973; Gimbrone et al., 1974) had

recently reported abundant proliferation of endothelial cells from human umbilical veins. Subsequently, many others described endothelial cell proliferation from other large vessels, such as aorta (Fenselau and Mello, 1976; Gospodarowicz, et af., 1977) and pulmonary artery (Ryan et al., 1978). Our result immediately suggested another important physiologic difference between capillary endothelial cells and pericytes in the retina, perhaps related to the by now well-known observation that, early in the course of diabetic retinopathy, pericytes are selectively lost from the retinal capillaries, while endothelial cells, at least initially, are preserved (Cogan et al., 1961; Yanoff, 1966; Speiser ef al., 1968). Biochemical studies of cultured pericytes could begin an experimental approach, both to an understanding of their physiology and function, and to the pathogenesis, at the molecular level, of disease processes such as diabetic retinopathy. Initial steps in this direction were taken with the report that cultured pericytes possessed aldose reductase activity (Buzney et al., 1977), an enzyme pathway known to be responsible for cataract formation in diabetic and galactosemic animals (Kinoshit~ 1965; Gabbay, 1973) and perhaps in man (Varma et at., 1979). It is possible that other complications of diabetes may also be attributed to this enzyme (Gabbay, 1973, 1975). In addition, we have recently found that cultured bovine retinal pericytes are capable of a limited amount of collagen biosynthesis (Cohen et ul., 1979). Further investigations, now in progress, are attempting to determine the types of collagen synthesized by these cells, and to learn whether collagen synthesis in retinal pericytes is affected by alterations in the glucose concentration of the culture medium, or by other chemical variations that may be related to disease states. For example, basement membrane biosynthesis in the rat renal glomerulus is altered in diabetes, a feature that may have a direct bearing on diabetic glomeruloscler165

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osis (Spiro and Spiru, 1971). Other investigations in our laboratory at the present time include studies, using isolated microvessels, of pathways of glucose metabolism in these vessels, as determined by (14C02) production using (6-14C)-D-glucose and (I- 14c)-Dglucose as substrates, and investigations of insulin receptors in cultured pericytes. An important problem in all this work is to relate the biochemical behavior of cultured cells to those in aivo. This can be done, but with some caution, by studying the biochemical properties of isolated microvessel fragments, as we are now doing in the experiments on pathways of glucose metabolism. The caution is necessary because it is at present impossible, without resorting to culture, to separate pericytes from endothelial cells in the vessel fragments. Recently, both Buzney and I, now working independently, have been able to culture retinal capillary endothelial cells (Buzney and Massicotte, 1979; Frank et at., 1979). The point of interest here is that retinal capillary endothelial cells proliferate only from microvessel fragments of fetal retinas, or from retinas whose capillary cells have not fully differentiated into mature pericytes and endothelial cells. Buzney has obtained endothelial cell proliferation from fetal calf retinas, while we have had success with fetal pigs and 34-week-old kittens and puppies. In our hands, these cells sometimes grow together with cells that have the appearance of pericytes, but sometimes only endothelial cells are observed in culture. Stranger still, on occasion the pericyte elements will selectively die, leaving only colonies of endothelial cells. Since we have not varied the culture conditions in these different experiments, the explanation for this behavior remains obscure. There are also species differences. In our experience, retinal endothelial cells from puppies (in two experiments) grew rapidly, formed monolayers that completely filled 35 mm culture dishes, and were successfully passed twice before they reached senescence. Endothelial cells from fetal pig and kitten retinas grow more slowly, and we have not yet had success in passing them. An obviously important question in these experiments is, how do we identify the cells we have cultured? For pericytes, the problem is somewhat diffi-

cult. Pericytes are identifiable in capillaries by their location in the vessel wall, outside the basement membrane of the endothelial cells, and, by light microscopy, by their small, round, darkly-staining nuclei. In culture, none of these features are present. They possess no specific organelles, and they have no known unique biochemical or physiological features. Hence, to date, our only means to identify the origin of these cells when we see them in culture is by radioautography of vessel fragments after a brief culture period (Buzney et al., 1975). In culture, they have a characteristic appearance (Fig. IS). Two other methods of identification suggest themselves. One would be immunologic. Cultures of pericytes could be used to produce antibodies in rabbits and, after absorption against various bovine tissues, indirect immunofluorescence or peroxidase techniques could be carried out using isolated microvessel fragments. A second method might rely on specific surface membrane glycoprotein receptor patterns. We have recently observed that. when cultured bovine retinal pericytes are incubated with SOpg/ml wheat germ agglutinin, multple vacuoles are observed in the cells within 2448 hr, although the cells remain viable for many days. The effect appears to be due to a specific carbohydrate binding site, because it is not present if the cells are pre-incubated with N-acetyl glucosamine, a specific “blocker” of the lectin. Other lectins, such as soybean agglutinin, ricin, and concanavalin A, do not cause similar vacuolization when added in the same concentrations. Ricin and soybean agglutinin are toxic, causing cell death within 24 hr. The vacuolization is similar to what Edelson and Cohn (1974a, b) have observed in cultured mouse peritoneal macrophages in the presence of concanavalin A. They demonstrated that this lectin stimulates pinocytosis, but no phagocytosis in cultured macrophages. It has been suggested (Michaelson cct al., 1974; Tripathi and Tripathi, 1978) that retinal capillary pericytes might be phagocytic cells, and the frequent observation of multiple mi~opinocytotic vesicles (Buzney et ni.. 1975) certainly indicates that this process is very active as well. Thus, we have begun to investigate whether wheat germ agglutinin stimulates pinocytosis in cultured pericytes, and whether they differ in this

Fig. 1. (A) Kitten retinal capillary endothelial cells following one month in cuiture. Phase contrast, x 200. (8) Bovine retinal capillary pericytes following two weeks in culture. The amorphous mass in the center of the photograph is the curled-up remnant of capillary basement membrane. From Frank et al (1979). Reproduced by permission of the editors.

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Fig. 2. Electron micrographs of cultured kitten retinal capillary endothelial cells, showing zonulae occludentes (“tight junctions”). (A) The arrow shows an endocytic vacuole. x 86,000. (B) Another “tight junction” between two cells, showing a typical lip-like protrusion. x 118,000. (C) A higher magnification of a function, showing a pentalaminar fusion zone. x 261,000. From Frank et al. (1979). Reproduced by permission of the editors.

respect from endothelial cells. However, the most important point in the present context is whether it is possible to demonstrate specific patterns of membrane Iabelling with wheat germ agglutjnin and other lectins that will identify cultured pericytes with their predecessors in the intact retinal capillary wall, and will differentiate them from endothelial cells. Concanavalin A readily lends itself to peroxidase histochemistry and electron microscope cytochemistry. Although the process is more difficult, similar labelling can be carried out with other lectins (Monsigny et af., 1976) enabling ready identification of membrane glycoprotein receptor sites. Identification of capillary endothelial cells is much easier. In culture, they have a characteristic morphology (Fig. lA), appearing as a mosaic of closely apposed, usually hexagonal cells. By electron microscopy, we have observed in kitten retinal pericytes zonulae occludentes (“tight junctions”, Fig. 2) some of which have a typical lip-like protrusion of the cytoplasm of the adjacent cells (Frank et al., 1979). Other endothelial cell “markers” may also be used. Buzney and Massicotte (1979) demonstrated factor VIII immunofluorescence in cultured capillary endothelium from fetal calf retinas. This technique has been somewhat more difficult for us, since there exists no feline

anti-factor VIII antibody, and cross-reactivity of cat plasma with the anti-human antibody is poor. However, we have recently observed a granular, perinuclear pattern of fluorescence in kitten retinal endothelial cells incubated with a 1:50 dilution of antihuman factor VIII (Boehringer). Pericytes cultured from kitten cerebral capillaries did not fluoresce. The reaction is not as impressive as that which has been demonstrated in other cultured endothelial cell lines from bovine and human tissues, however, A porcine anti-factor VIII antibody is available, but unfortunately there is little or no reactivity in cultured pig endothelial cells, despite a strong reaction with endothelial cells in vessels in freshly prepared frozen sections (D. Fass, personal communication). We are also planning to study immunofluorescence in cultured puppy endothelial cells using an anti-canine factor VIII antibody being prepared by Dr. David Fass of the Mayo Clinic. Still another endothelial cell marker is the presence of angiotensin converging enzyme activity. This is present in high concentrations in cultured aortic endothelial cells, but in lower concentrations in retinal capillary endothelium in culture. Once again, in our hands, there appeared to be species variations, with the highest activity appearing in cultured canine reti-

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nal capillary endothelial cells, and lesser activity in fetal pig and kitten celis. We observed no activity in cultured pericytes. The enzyme assay is a modification of a commer~ally available method (Ventrex Laboratories, Portland, Maine). The substrate is (3H)-hippuryl diglycine. The enzyme, a terminal dipeptidase, cleaves the terminal two glycine residues, leaving (“H)-hippuric acid. This is extractable in ethyl acetate. Hence, counting the ethyl acetate layer in a liquid scintillation counter quantitatively demonstrates activity. Demonstration that the activity is due to angiotensin converting enzyme is provided by the use of a specific inhibitor, SQ-20,881 (Teprotide), which has been kindly provided to us by the E. R. Squibb Co. The availability of cultures of retinal capillary pericytes and endothelial cells now provides us with a powerful method for the study of the biochemistry and function of the retinal vasculature in health and disease. Our investigations in this field are still in their early stages, but the future appears promising. il(~kno~~lecigemenrs--This investigation was supported by a research grant number EY-01857 from the National Eye Institute, National Institutes of Health, and by grants from the Juvenile Diabetes Foundation. I thank K. P. Mikus and Ann Randolph for technical assistance. REFERENCES

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