ceeding with a discussion of the biochemical basis of diabetic retinopathy.

Current Hypotheses for the Biochemical Basis of Diabetic Retinopathy LAWRENCE J. MANDARINO, PHD

Diabetic retinopathy is one of the leading causes of vision loss in industrialized countries. Despite recent advances, the biochemical basis for the development of this diabetic complication is uncertain. Although retinal circulation is unique in that it is readily observable noninvasively, retinal tissue is extremely difficult to study in humans because of the problems inherent in obtaining fresh, appropriate biopsy material. Moreover, because of the difficulties in working with animal models of diabetic retinopathy, such as the dog, many investigators have turned to cell-culture models, especially those using primary cultures of retinal capillary endothelial cells and pericytes. Diabetic retinopathy involves both morphological and functional changes in the retinal capillaries. Morphological changes include basement membrane thickening and pericyte disappearance; functional changes include one important early change—increased permeability—which may be attributable to endothelial cell changes and basement membrane leakiness. Investigators have described major biochemical changes in cellular signaling pathways, including myo-inositol, inositol phosphates, and DAG metabolism, as well as decreased Na+-K+-ATPase and increased PKC activity. These defects may be related to the way endothelial cells and pericytes synthesize and interact with the extracellular matrix. Abnormalities in endothelial cell or pericyte interaction with the basement membrane may in turn lead to functional abnormalities, such as increased permeability.

D

iabetic retinopathy remains one of the leading causes of vision loss and blindness in industrialized countries. Despite advances in laser photocoagulation therapy and its effectiveness in preventing or delaying the complications of diabetes that lead to blindness, the fact that the basis for this

FROM THE DEPARTMENTS

OF OPHTHALMOLOGY

therapy is only conjectural signifies that the understanding of the etiology of diabetic retinopathy is still minimal. This review, although not intended to be exhaustive, attempts a coalescence of several current lines of study. A brief review of the morphology and cell biology of retinal capillaries is necessary before pro-

AND PHYSIOLOGY,

PITTSBURGH; AND THE UNIVERSITY OF PITTSBURGH, PITTSBURGH,

THE EYE &

EAR INSTITUTE OF

PENNSYLVANIA.

ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO LAWRENCE J. MANDARINO, P H D , DEPARTMENT OF OPHTHALMOLOGY, THE EYE & EAR INSTITUTE OF PITTSBURGH, 203 LOTHROP STREET, PITTSBURGH, PA

15213. PKC,

PROTEIN KINASE Q

D A G , DIACYLGLYCEROL; R P E , RETINAL P1GMENTED EPITHELIUM; S T Z ,

STREPTOZOCIN; ALX, ALLOXAN; IDDM, INSULIN-DEPENDENT DIABETES MELLITUS; NIDDM, NON-INSULIND E P E N D E N T DIABETES MELLITUS.

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RETINAL MICROVESSEL STRUCTURE AND FUNCTION — Retinal circulation is unique in that it is readily visible, and can be observed easily and photographed noninvasively. However, by its nature, it is a difficult tissue to study in humans because of the problems inherent in obtaining fresh, appropriate biopsy material. Retinal capillaries are composed of endothelial cells that line the vessel, rest on a basement membrane, and are surrounded by pericytes, which are themselves embedded in basement membrane. The ratio of endothelial cells to pericytes is ~ 2 : 1 . In recent years, it has become possible to establish relatively pure cultures of retinal capillary endothelial cells and pericytes by using selective homogenization, purification, and culture techniques (1,2). Numerous sources for these cells have been used, but perhaps the most popular has been the bovine retina, because of its size and the ready availability of fresh tissue. Primary cultures of bovine retinal capillary pericytes and endothelial cells are shown in Fig. 1. CELLULAR AND MORPHOLOGICAL CHANGES CHARACTERISTIC OF DIABETIC RETINOPATHY— The endothelial cells that line the retinal capillary are interconnected by structures resembling tight junctions, and, together with the extracellular matrix components of the basement membrane, form a selective permeability barrier to the passage of large blood proteins and small molecules (such as fluorescein—commonly used as a diagnostic tool for studying retinal vascular disease). A reduced ability of the retinal microvasculature to perform its barrier/transport function can be one of the earliest functional changes in retinal circulation induced by diabetes. The function of the pericytes remains unknown, although it has been hypothe-

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Mandarino

Figure 1—Photomicrographs of primary cultures of bovine retinal capillary endothelial cells (A) and pericytes (B).

sized that they may be involved in contractility (3), may help maintain barrier/ transport function (4), or may help control proliferation of endothelial cells (5,6). By using scanning electron microscopy of ghosts of bovine retinal microvessels (basement membrane tubes

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from which cellular material has been removed by hypotonic and detergent lysis), it has been shown that pores exist in the retinal capillary basement membrane that would enable close contact between endothelial cells and pericytes (7). Thus, there appear to be anatomical avenues of

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communication that could allow for coordination of the maintenance of retinal capillary structure and function by endothelial cells and pericytes. Studies from the 1960s show that one of the most striking morphological features of human diabetic retinopathy is the selective disappearance of pericytes from retinal capillaries (8,9). Because this may precede some of the other vascular changes, such as neovascularization, it has been proposed that pericytes may prevent proliferation of retinal capillary endothelial cells. Orlidge and D'Amore (6) offered direct evidence of such a function for pericytes. These investigators cocultured retinal capillary endothelial cells with pericytes under two conditions: one in which physical contact between the two cell types was allowed to occur, and another in which the cell types were separated by a membrane that allowed diffusion of soluble materials but prevented direct cell contact. It was found that when direct contact between the pericytes and endothelial cells was allowed, the pericytes effectively inhibited endothelial cell proliferation (6). However, pericytes had no effect when direct contact was prevented. Interestingly, smooth muscle cells from aorta also were capable of preventing endothelial cell proliferation, adding evidence to the notion that capillary pericytes and smooth muscle cells from the walls of larger blood vessels may have common functions. Other cell types, such as fibroblasts or epithelial cells, actually had the reverse effect and increased endothelial cell proliferation in this system. Although the precise mechanism by which the pericytes inhibit proliferation is unclear, such a model system should be useful in its determination. OBSTACLES TO STUDY OF THE PATHOGENESIS OF DIABETIC RETINOPATHY— One factor that makes the study of diabetic retinopathy difficult is the relative inaccessibility of the human retinal vasculature. Although

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Biochemical basis of retinopathy

Table 1—Models for studying the biochemical basis of diabetic retinopathy MODEL STZ-INDUCED DIABETIC RAT

ADVANTAGES IN VIVO MODEL, EASY AND INEXPENSIVE TO MAINTAIN

ALX-INDUCED DIABETIC DOG

IN VIVO MODEL, RETINOPATHY SIMILAR TO HUMANS

GALACTOSEMIC RAT, DOG

DISADVANTAGES DISSIMILARITY TO SOME ASPECTS OF

REFERENCES 10,46,47,50

HUMAN DIABETIC RETINOPATHY EXPENSIVE AND DIFFICULT TO MAINTAIN

10-12

OVER LONG TERM NEEDED ( 3 - 5 YR) MAY REPRODUCE ONLY SOME ALTERATIONS

13-17

HUMAN TISSUE, EASY TO MANIPULATE

ENDOTHELIAL CELLS FROM LARGE BLOOD

29-32

VARIABLES IN TISSUE CULTURE, EASY

VESSELS MAY REACT DIFFERENTLY THAN

IN VIVO MODEL, EASIER TO MAINTAIN THAN DIABETIC ANIMALS

CULTURED HUMAN UMBILICAL VEIN ENDOTHEL1AL CELLS

TO MAINTAIN CULTURED BOVINE RETINAL CAPILLARY ENDOTHELIAL CELLS

CELL TYPES INVOLVED IN DIABETIC RETINOPATHY

THOSE FROM CAPILLARIES NONHUMAN CELLS MAY BEHAVE

1,2,18-28

DIFFERENTLY THAN HUMAN CELLS

AND PERICYTES

it is true that this is one of the few vascular beds that can be observed readily and noninvasively, obtaining biopsy or necropsy samples is difficult. Retinas from deceased donors often can be obtained only several hours after death occurred, and many biochemical alterations can occur during this time. In addition, many donor eyes are obtained from elderly individuals, and this can result in confusion between the effects of aging and those of diabetes. Moreover, it may not be easy to document such characteristics as glycemic control during the period of development of diabetic retinopathy years earlier. The following models for study of the mechanisms responsible for diabetic retinopathy are summarized in Table 1. A major impediment to understanding the biochemical basis of this complication is the lack of an easily workable animal model. Many animal models of diabetes have been characterized, but relatively few develop the morphological changes characteristic of diabetic retinopathy. The most widely used and easily accessible animal model of diabetes is the STZ-induced diabetic rat; but, unfortunately, the retinopathy that develops in this model is somewhat dissimilar to human diabetic retinopathy. Probably the most successful model of diabetic retinopathy is the ALX-induced

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diabetic dog (10). Numerous important cytes cultured from bovine eyes have studies have used this model and illus- been used to obtain data regarding glutrate its utility (10-12). The retinal mi- cose metabolism and insulin action ( 1 8 crovasculature of the dogs in these stud- 20), extracellular matrix protein synies develops pericyte loss, acellular thesis (21), regulation of vascular capillaries, saclike microaneurysms, and permeability (22), glucose transport acvessel tortuosity (11). Unfortunately, the tivity (23-25), analysis of the sorbitol similarity between this retinopathy and pathway (26,27), and PKC regulation the complication in humans extends to and DAG metabolism (28). One limitathe years it takes to develop in the dia- tion of this cell-culture model is that, betic dog. It is exceedingly difficult and because the cells are not human, their expensive to maintain these animals for behavior may differ in species-specific time necessary to gain needed data. Re- ways. Furthermore, long-term passage cently, high galactose feeding has been and maintenance of the cells is not feaused to produce a diabeticlike retinopa- sible currently, which limits experimenthy in rats (13-15) and dogs (16,17), tal designs. and the retinopathy is similar to that in To avoid these limitations, some diabetic dogs (16). Assuming that the investigators have used endothelial cells pathogenesis of galactosemia-associated cultured from human umbilical veins retinopathy is the same as diabetic reti- (29-32). These cells have been used to nopathy, this model may prove useful. gain valuable information about the efWith the lack of accessibility of fects of elevated glucose concentrations human tissue samples and the expense on replication (29), DNA damage (29), and difficulty of using a good animal and extracellular matrix synthesis (30model of diabetic retinopathy, many in- 32). These cells carry the advantages that vestigators have turned to cell-culture they are of human origin and are culmodels, which use a variety of relevant tured relatively easily from a readily obcell types. It is now possible to establish tainable source. One disadvantage is that relatively pure primary cultures of endo- endothelial cells from major blood vesthelial cells and pericytes from retinal sels may not react to diabeticlike condimicrovessels (1,2). Most investigators tions in the same way as endothelial cells have used retinas from bovine eyes be- from microvessels. This is because the in cause of their size and the accessibility of vivo lesions typical of retinal microvesfresh tissue. Endothelial cells and peri- sels in diabetes are different from those

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that affect endothelial cells in major blood vessels, such as an increased occurrence of atherosclerotic lesions. ELEVATED GLUCOSE CONCENTRATION AND DEVELOPMENT OF RETINOPATHY— Despite intensive study, the precise biochemical and molecular mechanisms responsible for the development of diabetic retinopathy remain uncertain. Because retinopathy affects patients with either IDDM or NIDDM, it is widely believed that the factor responsible for its development must be a symptom common to both diseases, although they have widely different etiologies. Although the most logical candidate for a causative factor is hyperglycemia, the link has been difficult to establish with a high degree of certainty. Many studies of the epidemiology and risk factors of diabetic retinopathy have shown a positive correlation between the severity of hyperglycemia and the degree of diabetic retinopathy ( 3 3 35). Because these types of studies reveal associations between variables, not causation, clinical trials have been undertaken to determine whether improvement in glycemic control impedes the progress of retinopathy (36-40). The findings of these studies have been somewhat mixed, although current results suggest a beneficial effect of good glycemic control if it is maintained for at least 3—4 yr. In fact, some of these studies have reported transient worsening of retinopathy after 1-2 yr of tight glycemic control (41). The largest of such studies, the Diabetes Control and Complications Trial, is still underway (40). Part of the difficulty in establishing a direct link between glycemic control and retinopathy has been the lack of a good animal model in which to test the hypothesis that tight control of blood glucose concentrations slows the progression of retinopathy. The development of the ALX-induced diabetic dog model (10) has allowed such an experi-

DIABETES CARE, VOLUME 15,

ment to be conducted. In what has become a classic experiment, Engerman and Kern (11) induced diabetes with ALX in mongrel dogs and divided the animals into three groups. One group was allowed to remain under poor glycemic control for 5 yr; one group was maintained under strict glycemic control for 5 yr, with multiple daily insulin injections; and one group was allowed to remain under poor glucose control for 2.5 yr, and then was switched to strict control for another 2.5 yr. At 2.5 yr, one eye of each dog was removed, and the retinal vasculature was analyzed histologically. The other eye was examined after 5 yr (at the completion of the experiment). A group of nondiabetic dogs served as controls. No significant retinopathy was observed in any group after 2.5 yr. However, after 5 yr, the group under poor control evidenced significant retinopathy, including microaneurysms, pericyte loss, and acellular capillaries; whereas the group under strict control for 5 yr showed no more retinopathy than nondiabetic dogs. The group that was switched from poor to strict control surprisingly had a similar amount of retinopathy as the group under poor control for all 5 yr. This suggests that a process independent of gross morphological changes, once started, may proceed inexorably, regardless of subsequent strict glycemic control. Further evidence for this concept comes from studies of pancreas transplant recipients at the University of Minnesota (42). A group of patients with successful pancreas transplants (as judged by their withdrawal from insulin injections) was compared with a group of diabetic patients with failed pancreas grafts. No difference in progression of retinopathy was noted between the groups, and, in fact, one patient with a successful transplant subsequently developed proliferative diabetic retinopathy, even though blood glucose was normalized. Finally, although these studies in dogs clearly establish that tight glycemic control can prevent or delay the appear-

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ance of retinopathy, they do not prove that glucose itself is the causative factor because many other metabolic and hormonal parameters are normalized when euglycemia is well-maintained. Other studies, with rats (15) and dogs (16,17), have demonstrated that induction of galactosemia, the result of feeding animals a high-galactose diet, produces a retinopathy that is similar to the retinopathy produced by diabetes in these animals. Galactosemia is unaccompanied by the other metabolic changes that characterize diabetes mellitus, so it can be concluded that elevated galactose concentrations probably cause this retinopathy. These findings make it highly likely that increased blood concentrations of hexose are the primary causative factor in the pathogenesis of diabetic retinopathy. It is therefore important to give consideration to hypotheses regarding the biochemical basis of this complication that start with the premise that high glucose concentrations are responsible for initiating the chain of events that leads to the clinically significant lesions of diabetic retinopathy. CURRENT HYPOTHESES OF THE BIOCHEMICAL BASIS OF DIABETIC RETINOPATHY

Sorbitol pathway Possibly the most widely studied biochemical pathway that has been proposed to be responsible for diabetic complications is the sorbitol (polyol) pathway. In this pathway, a hexose, such as glucose or galactose, is converted by aldose reductase to its respective sugar alcohol, sorbitol, or galactitol. An important property of sugar alcohols is that they do not pass readily through cell membranes, so they can accumulate to high levels intracellularly. In many tissues, sorbitol dehydrogenase can convert sorbitol to fructose, which can be metabolized and thus decrease sorbitol concentrations. This enzyme is relatively inactive and uses galactitol as a substrate, however, intracellular galactitol concen-

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Biochemical basis of retinopathy

trations can achieve extremely high levels. This is the basis for the concept of using galactose feeding in dogs or rats to mimic diabetic complications potentially related to the sorbitol pathway in diabetes. In its original formulation, this hypothesis was intended to account for cataract formation in the ocular lenses of diabetic humans and animal models (43). The presence of aldose reductase activity and high levels of sorbitol have been demonstrated in the lenses of diabetic animals (43,44), and it has been shown that many of the changes leading to these cataracts likely were attributable to hyperostotic effects of sorbitol or galactitol accumulation in the lens (43). This early formulation of the way in which the sorbitol pathway might be involved in producing diabetic complications, although apparently responsible for diabetic cataracts, by itself could not account for diabetic complications in other tissues because the concentration of sorbitol did not reach levels high enough to produce significant changes in osmolarity (45,46). This led to a reexamination of how the sorbitol pathway was involved in diabetic complications. Studies on the pathogenesis of diabetic neuropathy had shown that increased polyol pathway activity led to decreased nerve myo-inositol concentrations and decreased motor nerve conductance velocity (47). It was suggested that decreased myo-inositol levels would result in decreased inositol phosphate concentrations, which could in turn lead to decreased DAG concentrations. The fall in DAG might then lead to decreased Na + K+-ATPase activity, decreasing the normal Na + gradient required for conduction of an action potential by neurons (48). More recently, it has been suggested that this formulation may apply to the RPE, which actively transports materials out of the retinal extracellular fluid (49). A decrease in transport of fluorescein out of the retina may be an early functional change in the development of

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diabetic retinopathy (50,51); and it has been shown that in experimental diabetes in rabbits, RPE sorbitol concentrations were increased, and myo-inositol concentrations and Na+-K+-ATPase activities were decreased (52,53). Furthermore, the Na + gradient across the RPE layer was diminished in the diabetic eyes, indicating the functional consequences of decreased Na+-K+-ATPase activity. Whether these mechanisms are involved in the pathology of retinal capillaries in diabetes is not clear. Aldose reductase is presumed to be present in retinal capillary cells, based mainly on findings that aldose reductase inhibitors prevent sorbitol or galactitol accumulation in these cells (54,55), so the potential exists for the involvement of this pathway. However, immunostaining failed to reveal the presence of the enzyme in retinal capillaries (56). Culture of bovine retinal capillary endothelial cells in the presence of high glucose concentrations increases cell sorbitol concentrations and decreases Na + -K + ATPase activity (28), as in neurons, and these changes are preventable by sorbinil, an aldose reductase inhibitor. In contrast to nerve tissue, however, myoinositol or inositol phosphate levels in the endothelial cells were unchanged by the high-glucose treatment (28). Nevertheless, the possibility remains of a decrease in myo-inositol in a small, rapidly turning over discrete pool (49), which might not have been measurable by the techniques used in the endothelial cell study. The diabetic and galactosemic dog and rat models of retinal microvascular changes have been examined for the ability of various aldose reductase inhibitors to prevent or delay the onset of these changes. Because the galactosemia model presumably works by means of polyol pathway involvement, prevention of pathological changes by aldose reductase inhibitors would be strong evidence of the importance of this pathway in the etiology of retinopathy. Studies of this

kind have been conducted by two groups. Kador et al. (17) and Robison et al. (13-15), using both dog and rat models and various aldose reductase inhibitors, found a pronounced ability of these inhibitors to delay or prevent the appearance of microvascular abnormalities. In direct opposition to these results, Kern and Engerman (56), using either diabetic or galactosemic dogs and sorbinil, found that sorbinil, given at a dose that inhibited erythrocyte sorbitol accumulation by 80%, did not prevent the occurrence of retinal microvascular abnormalities. Moreover, although studies are still being conducted, little or no data has accumulated to suggest that aldose reductase inhibitors have a marked beneficial effect on the progression of retinopathy in human diabetic patients. In cultured retinal capillary pericytes, some intriguing data demonstrate the various effects of high glucose levels on myo-inositol and inositol phosphate metabolism. Li et al. (26) showed that either glucose or galactose concentrations > 5 mM inhibited myo-inositol transport into pericytes, but the nonmetabolizable hexoses, L-glucose and 3-0methylglucose, were without effect. Moreover, the inhibition of myo-inositol transport was at least partially reversible by sorbinil. In another study, Li et al. (27) showed that in the presence of high glucose, pericytes convened less myoinositol to inositol triphosphate, and concluded that high glucose therefore may interfere with normal intracellular signal transduction by this mechanism. Taken as a whole, a good deal of evidence suggests that the sorbitol pathway, through its effect on myo-inositol metabolism, plays an important role in some of the functional changes observed in diabetic complications in general and in retinopathy in particular. Nevertheless, contradictory data are sufficient to indicate that this may not be the only or even primary biochemical mechanism responsible for the development of retinopathy.

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Effect of glucose on PKC activity The current concepts of the effects of increased sorbitol pathway activity on myo-inositol concentrations and turnover of inositol phosphates opens the possibility that high glucose might alter PKC activities. If cellular DAG concentrations were decreased, a decrease in PKC activity would be predicted. Because PKC activity is involved in the regulation of many cellular functions (58), such an effect could have pleiotropic implications for the cell. Numerous researchers have reported that elevated glucose concentrations in vivo or in vitro result in an increase, rather than a decrease, of PKC activity in the kidney (59), the heart (60), retinal capillary endothelial cells (28), and skin chamber granulation tissue (61). This effect has been especially well-characterized in the skin chamber granulation tissue model (61). This model allows the assessment of the effects of glucose on microvascular function, specifically blood flow and albumin permeability of the vasculature, and direct measurement of biochemical parameters on the involved tissues. The investigators have shown that instillation of high-glucose solutions into the granulation chambers increases blood flow and albumin permeability—functional changes that mirror those in diabetes. Moreover, glucose instillation resulted in a near threefold increase in DAG concentrations, which is compatible with the increased PKC activity reported in microvascular cells in diabetes (28), but not consistent with predictions of the sorbitol pathway hypothesis. These investigators also showed that pharmacological manipulations that would increase or decrease DAG in the granulation tissue resulted in the expected accompanying changes in microvascular function (61). Lee et al. (28) performed studies in bovine retinal capillary endothelial cells that addressed the question of the effects of glucose on PKC activity. This team found that when these cells were exposed to high glucose, Na + -K + -

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ATPase activity was decreased, as expected from the sorbitol/myo-inositol hypothesis, but PKC activity was increased twofold in a membranous pool. Although the decrease in Na+-K+-ATPase activity was prevented by sorbinil, this aldose reductase inhibitor had no effect on the increase in PKC activity. These data suggest that changes in sorbitol pathway activity and PKC activity may be induced by glucose via different biochemical mechanisms. The sorbitol/jriyo-inositol hypothesis would predict that decreased myo-inositol concentrations might result in decreased DAG concentrations, which would lead to decreased, not increased, PKC activity. Thus, the documented increase in PKC activity in diabetes seemingly is incompatible with the sorbitol/ myo-inositol hypothesis. However, it may be that rnyo-inositol concentrations do not reflect myo-inositol turnover rates, and it is conceivable that an increased myo-inositol turnover rate could lead to increased DAG levels. It certainly is possible that turnover rates of a substrate can be increased, even though its concentrations are decreased. The importance of PKC in the regulation of cellular activities indicates that further investigation of this abnormality should receive high priority. Pathogenesis of basement membrane abnormalities in diabetic retinopathy Thickening of capillary basement membranes in general and the retinal capillaries in particular has long been considered a hallmark morphological characteristic of diabetic microvascular disease. Although this abnormality may be predated by functional changes in the capillaries, basement membrane expansion could be the result of ongoing abnormalities in extracellular matrix synthesis and recognition by retinal capillary endothelial cells and pericytes. It is widely thought that normal interactions of cells with their basement membrane are required for their normal function

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(62,63). Defects in recognition of basement membrane by retinal capillary endothelial cells could at the same time be responsible for functional abnormalities, such as increased permeability and the development of thickened basement membrane, in an effort to compensate for the perceived abnormality. Recognition of extracellular matrix proteins In most tissues, microvascular basement membranes are composed of type IV collagen, laminin, basement membrane (heparan sulfate) proteoglycans, and entactin, which presumably are secreted and deposited by endothelial cells, pericytes, and fibronectin, which is secreted and deposited by microvessel cells in addition to being deposited from circulation. Of these components, laminin, fibronectin, and collagen IV potentially are involved in attachment of microvessel cells to the extracellular matrix. In assays in which plastic culture dishes were precoated with purified basement membrane components, both laminin and fi~ bronectin have been shown to be capable of promoting the attachment of bovine retinal capillary endothelial cells and pericytes in a dose- and concentrationdependent manner (Fig. 2). Heparan sulfate proteoglycan was incapable of promoting cell attachment (64). To determine whether high glucose concentrations could alter the interaction of endothelial cells with basement membrane, retinal capillary endothelial cells were cultured in the presence of 5 or 20 mM glucose for 4 days before determination of their subsequent ability to reattach to laminin or fibronectin. The high glucose concentration inhibited the attachment of the endothelial cells to laminin by 20% but had no effect on attachment to fibronectin (65). This suggests that a brief (4-day) exposure to a glucose concentration (20 mM) similar to that observed in poorly controlled diabetic patients might induce changes in recognition of basement membrane components well before any morphological

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High (40 mM) glucose concentrations induce an increase in total collagen synthesis by cultured bovine retinal capillary pericytes (66), as assessed by incorporation of radiolabeled proline. Using cultured human umbilical vein endothelial cells, Cagliero et al. (31) have shown that high glucose increased fibronectin and type IV collagen mRNA levels, protein content, and vitamin B- chain mRNA. The increased mRNA levels were attributable to increased tran- Figure 3—Model for the interaction of abnorscription. Although these studies were malities in intracellular signaling with synthesis performed in cultured cells, nonhuman and recognition of extracellular matrix proteins. cells, or cells from major blood vessels (FN), Fibronectin; (LN), laminin; (HSPG), rather than capillaries, they nevertheless heparan sulphate proteoglycan; shaded area repoffer intriguing data. A major advance in resents the basement membrane; box above it this area would be the development of represents a retinal capillary endothelial cell. methods to address these questions in human retinal capillary cells in vitro and lar matrix by retinal capillary cells could in vivo. lead to changes in synthesis of basement membrane components as a compensaMODEL FOR THE PATHOGENESIS tory effort. Moreover, changes in PKC or OF DIABETIC RETINAL other cellular regulatory factors conceivMICROVASCULAR DISEASE — ably could alter basement membrane Given the above evidence, a model for synthesis or recognition. For example, the early development of biochemical endothelial cell proliferation and phenochanges in diabetic retinopathy may be type is strongly regulated by the extradeveloped (Fig. 3). cellular matrix (70,71). Altered interacFirst, growing evidence suggests tions of retinal capillary endothelial cells that elevated glucose concentrations alter with their basement membrane could rethe activities and concentrations of cel- sult in the principal early abnormality in lular signaling systems, including inosi- retinopathy— capillary leakiness—and tol phosphates, DAG and cellular Ca 2+ even lead to the marked proliferative levels, Na+-K+-ATPase, and PKC. It is changes observed in the late stages of this known that PKC, through phosphoryla- disease. tion of the insulin receptor, is capable of altering its turnover and, potentially, its SUMMARY— Current hypotheses for function (67). If changes in PKC activity the biochemical basis of diabetic retinopalso affected the function of cell-surface athy center around potential abnormalireceptors, such as the integrins or other ties in intracellular signaling pathways. receptors for extracellular matrix compoAlthough data are still meager, it is likely nents, this could alter the manner in that defects in these pathways could rewhich the cells perceive their basement sult in the functional and morphological membrane. Moreover, integrin-mediated changes observed in diabetic retinopathy cell attachment to the extracellular mavia a mechanism involving changes in trix has been reported to be increased by cell recognition and synthesis of extracelphorbol ester treatment (68). In addilular matrix components. 2+ tion, some evidence suggests that Ca is a physiological regulator of integrinmediated cell attachment (69). A percep- Acknowledgments—This work was suption of an abnormality of the extracellu- ported by the Juvenile Diabetes Foundation, QLUCOSE GALACTOSE

1 NA-K-ATPase T PROTEIN K I N A S E C T DIACYLGLYCEBOt

/FN. I

40

60

80

100

120

FIBRONECTIN (uG/ml)

THICKENED BASEMENT MEMBRANE. FUNCTIONAL CHANGES IN CELL-CELL AND CELL-MATRIX INTERACTON LEADING TO CAPILLARY LEAKINESS

40

60

80

100

120

LAMININ (uG/mL)

Figure 2—Characteristics of attachment of bovine retinal capillary endothelial cells and pericytes to fibronectin (A) and laminin (B). Cells were labeled overnight with [3H]thymidine, trypsinized, and passaged to plastic dishes that had been precoated with 0-100 g/ml of fibronectin or laminin. Data are expressed as the percentage of total cell-associated counts that had attached to the dishes afier 3 h. Fibronectin was from bovine plasma, and laminin was isolated from mouse Engelbreth-Holm-Swanson (EHS) tumor.

changes could be observed—and contemporaneously with changes in PKC activity (28).

Synthesis of basement membrane components Besides interacting with basement membrane components through specific receptors, retinal capillary endothelial cells and pericytes in culture are capable of synthesizing collagens (66), basement membrane proteoglycans and laminin (64), and fibronectin (J. Finlayson, L.J.M., unpublished observations). Alterations in rates of synthesis or turnover of these components theoretically could alter the morphology of the capillary basement membrane.

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National Institutes of Health Grant RO1EYO8391, The Pennsylvania Lions, Research to Prevent Blindness, and The Eye and Ear Institute of Pittsburgh. The expert technical assistance of J. Finlayson and excellent editorial assistance of J. Smith are gratefully acknowledged.

References 1. Bowman PD, Betz AL, Goldstein GW: Primary culture of microvascular endothelial cells from bovine retina: selective growth using fibronectin coated substrate and plasma derived serum. In Vitro 18:626-32, 1982 2. GitlinJD, D'Amore PA: Culture of retinal capillary cells using selective growth media. Microvasc Res 26:74-80, 1983 3. Tilton R, Kilo C, Williamson J, Murch W: Differences in pericyte contractile function in rat cardiac and skeletal muscle microvasculartures. Microvasc Res 18: 336-52, 1979 4. DeOliveira F: Pericytes in diabetic retinopathy. BrJ Ophihalmol 50:134-43,1966 5. Kuwabara T, Cogan D: Retinal vascular patterns: VI. Mural cells of the retinal capillaries. Arch Ophthalmol 69:492502, 1963 6. Orlidge A, D'Amore PA: Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells. J Cell Biol 105:1455-62, 1987 7. Carlson E: Topographical specificity in isolated retinal capillary basement membranes: a high-resolution scanning electron microscope analysis. Microvasc Res 35:221-35, 1988 8. Cogan DG, Toussaint D, Kuwabara T: Retinal vascular patterns: IV. Diabetic retinopathy. Arch Ophthalmol 66:36678, 1961 9. Speiser P, Gittelsohn AM, Patz A: Studies on diabetic retinopathy: III. Influence of diabetes on intramural pericytes. Arch Ophthalmol 80:332-37, 1968 10. Engerman R: Animal models of diabetic retinopathy. Trans Am Acad Ophthalmol Oto-Laryngol 81:710-15, 1976 11. Engerman RL, Kern TS: Progression of incipient diabetic retinopathy during good glycemic control. Diabetes 36:808— 12, 1987

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