A monoclonal antibody (3G5)-defined ganglioside antigen is expressed on the cell surface of microvascular pericytes.

The identification of microvascular pericytes in vitro relies principally on morphological characteristics and growth dynamics, as there is a paucity of immunochemical markers for these cells. Consequently, an attempt was made to identify mAb reagents that would aid in both the rapid identification and enrichment of retinal capillary pericytes in vascular cell cultures. A panel of mAbs raised by xenogeneic immunization of mice with various tissues was screened for immunoreactivity with dissociated cultures of bovine retinal capillary pericytes. Two antibodies from the panel (3G5 and HISL-8) were seen to react with pericytes by indirect immunofluorescence. The mAb 3G5 was selected for further study. mAb 3G5 did not react with dissociated cultures of smooth muscle cells, endothelial cells, or retinal pigmented endothelial cells. The pericyte 3G5 antigen was insensitive to the action of trypsin; therefore, mAb 3G5 was used to selectively purify pericytes from trypsinized mixed retinal cell cultures by flow cytometry. 3G5+ pericytes (representing 8% of cells in a mixed retinal cell culture) were enriched at least nine-fold to represent greater than 70% of cells. The mAb 3G5 stained retinal capillaries in vivo with a fluorescence distribution consistent with pericyte staining. The 3G5 antigen of cultured pericytes was found to be a glycolipid of mobility intermediate between ganglioside markers GM1 and GM2.

The pathway between hyaloid blood and retinal neurons in the toad. Structural observations and permeability to tracer substances.

The hyaloid vessels form a capillary network on the inner surface of the retina. These capillaries are embedded in the vitreous humor, and they lack a glial investment. The intercellular spaces of the retina communicate with the ocular cavity, as can be evidenced by following the penetration of tracer substances. Hence, there is an extracellular diffusion pathway between hyaloid capillaries and retinal neurons, without interposition of glial cells. Trypan blue and ferrocyanide were not detected within the vitreous humor nor the retina after systemic injection. To this extent, at least, the hyaloid capillaries functionally resemble central nervous system capillaries. Intravascular injections of horseradish peroxidase established the absence of vesicular transfer across the endothelium of the hyaloid capillaries. In addition, quintuple-layered junctions between endothelial cells prevented the intercellular passage of the enzyme. It is likely, therefore, that the only pathway across the endothelium of the hyaloid capillaries is through the plasmalemma of the endothelial cells.

Functional sorting of actin isoforms in microvascular pericytes.

We characterized the form and distribution of muscle and nonmuscle actin within retinal pericytes. Antibodies with demonstrable specificities for the actin isoforms were used in localization and immunoprecipitation experiments to identify those cellular domains that were enriched or deficient in one or several actin isoforms. Living pericyte behavior was monitored with phase-contract video microscopy before fixation to identify those cellular areas that might preferentially be stained with either of the fluorescent antiactins or phallotoxins. Antibody and phallotoxin staining of pericytes revealed that nonmuscle actin is present within membrane ruffles, pseudopods, and stress fibers. In contrast, muscle actin could be convincingly localized in stress fibers, but not within specific motile areas of pericyte cytoplasm. To confirm and quantitatively extend the results obtained by fluorescence microscopy, nonionic and ionic detergents were used to selectively extract the motile or immobilized (stress fiber-containing) regions of biosynthetically labeled pericyte cytoplasm. Immunoprecipitated actins that were present within these discrete cellular domains were subjected to isoelectric focusing in urea-polyacrylamide gels before fluorographic analysis. Scanning laser densitometry of the focused actins could not reveal any detectable alpha-actin within those beta- and gamma-actin-enriched motile regions extracted with nonionic detergents. Moreover, when pericyte stress fibers are completely dissolved by ionic detergent lysis, three actin isoforms can be quantified to be present in a ratio of 1:2.75:3 (alpha:beta:gamma). These biochemical findings on biosynthetically labeled and immunoprecipitated pericyte actins confirm the fluorescent localization studies. While the regulatory events governing this actin sorting are unknown, it seems possible that such events may play important roles in controlling cell shape, adhesion, or the promotion of localized cell spreading.

Differential regulation of protein kinase C and (Na,K)-adenosine triphosphatase activities by elevated glucose levels in retinal capillary endothelial cells.

Elevated cellular sorbitol levels resulting from conversion of increased glucose by aldose reductase might deplete cellular myoinositol content, which could then lower inositol phosphates (InsPs) and diacylglycerol levels, key regulators of protein kinase C (PKC). Secondary to altered PKC activity, other cellular enzymes such as (Na,K)-ATPase could be affected. To test this hypothesis we examined the association between PKC activity, (Na,K)-ATPase activity, and sorbitol, myoinositol, and InsP levels in cultured bovine retinal capillary endothelial cells, a cell type prominently involved in diabetic retinopathy. Elevating glucose concentration in culture media from 100 to 400 mg/dl led to a 100% increase in sorbitol levels, which could be inhibited completely by sorbinil, an aldose reductase inhibitor. In contrast, no changes were observed in myoinositol or InsP levels. Subfractionated PKC activities showed a 100% increase in the membranous pool with a parallel decrease in the cytosolic fraction. Adding sorbinil did not affect PKC activity, whereas the PKC agonist, phorbol myristate acetate (PMA), stimulated translocation of PKC. Ouabain-inhibitable (Na,K)-ATPase activity was decreased 70% by elevated glucose levels. This decrease could be prevented by adding either PMA or sorbinil. Thus, in retinal capillary endothelial cells elevated glucose concentration can affect PKC and (Na,K)-ATPase activities, probably via different mechanisms.

Analysis of glycosaminoglycans in bovine retinal microvessel basement membrane.

Glycosaminoglycans (GAG) were isolated from bovine retinal microvessel basement membrane (RMV-BM) and quantitatively analyzed using a recently described competitive binding assay that is specific for and sensitive to nanogram amounts of heparan and chondroitin sulfates. Treatment of osmotically lysed retinal microvessels with the ionic detergent deoxycholate (DOC), required for liberation of the extracellular matrix for plasma membrane lipoproteins and purification of the insoluble matrix, solubilized less than 5% of the GAG in the water-insoluble material. Total GAG content in the DOC-insoluble basement membranes was approx. 0.52 micrograms/mg dry weight; about 70% of the measurable GAG was resistant to both chondroitinase ABC and chondroitinase AC digestion and was sensitive to nitrous acid treatment, indicating its heparan sulfate nature. Cellulose acetate electrophoresis revealed two bands, one of which had an electrophoretic mobility similar to heparan sulfate standard and was sensitive to nitrous acid; the other migrated in the same position as chondroitin sulfate standard and was sensitive to chondroitinase ABC and chondroitinase AC digestion. These results provide evidence that RMV-BM contains chondroitin sulfate(s) as well as heparan sulfate, and offer the first quantitative analysis of GAG in this extracellular matrix.

Presence of glycosaminoglycans in retinal capillary basement membrane.

Retinal microvessels were isolated from bovine eyes and the basement membranes were purified either directly or after incubation with [35S]sulfate and [14C]glucosamine. The basement membranes, which were purified by osmotic lysis and sequential treatment with detergents, had the general compositional features associated with basement membrane collagens, including high levels of hydroxyproline and hydroxylysine and the presence of 3-hydroxyproline and cystine. After pronase digestion, cellulose acetate electrophoresis of glycosaminoglycans from retinal microvessel basement membrane revealed material comigrating with heparan sulfate that was insensitive to digestion with Streptomyces hyaluronidase ad chondroitinase ABC. Retinal microvessels also incorporated [35S]- and [14C]glucosamine into glycosaminoglycans that were isolated following pronase digestion of the retinal microvessel basement membrane purified from these incubations. The findings provide the first demonstration that glycosaminoglycans are integral components of the retinal microvascular basement membrane and suggest that heparan sulfate is the major glycosaminoglycan species in this basement membrane.

Non-competitive inhibition of myo-inositol transport in cultured bovine retinal capillary pericytes by glucose and reversal by Sorbinil.

myo-Inositol transport by retinal capillary pericytes in culture was characterized. The major myo-inositol transport process was sodium-dependent, ouabain-sensitive, and saturable at 40 mM, indicating a carrier-mediated process. The sodium ion concentration required to produce one-half the maximal rate of myo-inositol uptake ([Na+]0.5) did not show dependence on the external myo-inositol concentration (22.3 mM sodium for 0.005 mM myo-inositol; 18.2 mM sodium for 0.05 mM myo-inositol). myo-Inositol transport was an energy-dependent, active process functioning against a myo-inositol concentration gradient. The kinetics of the sodium-dependent system fitted a 'velocity type' co-transport model where binding of sodium ion to the carrier increased the velocity (Vmax 28 to 313 pmol myo-inositol/micrograms DNA per 20 min when [Na+] varied from 9 to 150 mM) but not the affinity for myo-inositol (Km 0.92 to 0.83 mM when [Na+] varied from 9 to 150 mM). Metabolizable hexoses (D-glucose or D-galactose; greater than 5 mM) inhibited myo-inositol uptake. Dixon-plot analysis indicated that the inhibition was non-competitive with a Ki of 22.7 mM for D-glucose and 72.6 mM for D-galactose. The inhibition was significantly reversed by Sorbinil (0.1 mM), an aldose reductase inhibitor. In contrast, high concentrations of non-metabolizable hexoses (L-glucose, 3-O-methyl-D-glucose), or partially metabolizable 2-deoxy-D-glucose, did not significantly inhibit myo-inositol uptake. The inhibitory effect of D-glucose or D-galactose on myo-inositol transport appeared to be related to glucose or galactose metabolism via the polyol pathway.

Quantitative vitreous fluorophotometry. A sensitive technique for measuring early breakdown of the blood-retinal barrier in young diabetic patients.

Vitreous and aqueous humor fluorescein concentrations were measured one hour after graded intravenous fluorescein was given to 20 juvenile diabetics, ages 20 to 40, with and without retinopathy, and to 12 controls of similar age. Vitreous fluorescein concentrations were significantly higher in diabetics, indicating breakdown of the blood-retinal barrier. Mean vitreous fluorescein values were 10.66 +/- 0.65 for the diabetics and 4.28 +/- 0.37 ng./ml. for the controls. Breakdown of the blood-retinal barrier was also confirmed in diabetics under the age of 20 without retinopathy. The blood-aqueous barrier was similarly altered in diabetics. Vitreous fluorophotometry quantitatively measures breakdown of the blood-retinal barrier, possibly the earliest detectable ocular vascular abnormality in juvenile diabetic patients.

Progression of incipient diabetic retinopathy during good glycemic control.

To assess the extent to which the progression of diabetic retinopathy can be arrested by improved glycemic control, 35 normal dogs were randomly divided into a nondiabetic and three alloxan-induced diabetic groups prospectively identified according to glycemic control: poor control for 5 yr (PC), good control for 5 yr (GC), and poor control for 2.5 yr followed by good control for 2.5 yr (PGC). To achieve good control, insulin was given twice daily together with a measured diet so that hyperglycemia and glucosuria were mild and infrequent, and HbA1 was comparable to normal. Retinal capillary aneurysms and other lesions developed during 60 mo of poor control (group PC) and were inhibited if good control was begun promptly within 2 mo (group GC). In group PGC, retinopathy was absent or equivocal at 2.5 yr of poor control and, surprisingly, was found to develop subsequently despite good glycemic control. Retinopathy in group PGC was greater at autopsy than at 2.5 yr and was greater than in group GC. The results indicate that retinopathy may be preventable but tends to resist arrest even in its incipient stages, before more than the first few aneurysms have appeared.

Studies on the pathophysiology of diabetic retinopathy. The blood-retinal barrier in diabetes.

A review of the pathologic picture of diabetic retinopathy shows that available clinical methods of examination demonstrate the alteration of the blood-retinal barrier (leakage), microaneurysms, capillary closure, preferential channels, preretinal neovascularization and gross extravascular lesions. All of these changes may be shown by fluorescein angiography. The value of this method, however, is fundamentally related to the morphologic demonstration of these lesions and not their quantification. Quantitative evaluation of retinal involvement in diabetes is needed in order to delineate more clearly its natural history, criteria for prognosis, and effect of treatment. Vitreous fluorophotometry, a quantitative and sensitive method of evaluating the permeability of the blood-retinal barrier, has opened new perspectives for the evaluation of retinal involvement in diabetes. Vitreous fluorophotometry has shown that a disturbance of the blood-retinal barrier, possibly functional, appears in diabetic eyes before any lesion is clinically visible in the fundus, and that there is a close correlation between the severity of the vascular lesions and higher vitreous fluorophotometry readings. Recent studies also indicate an interesting correlation with metabolic control, particularly, glycosylated hemoglobin levels and insulin treatment. Finally, on the basis of these findings a working hypothesis for the pathogenesis of diabetic retinopathy is presented.

Increased vascular permeability in spontaneously diabetic BB/W rats and in rats with mild versus severe streptozocin-induced diabetes. Prevention by aldose reductase inhibitors and castration.

125I-labeled albumin permeation (IAP) has been assessed in various tissues in spontaneously diabetic insulin-dependent female BB/W rats and in male Sprague-Dawley rats with severe or mild forms of streptozocin-induced diabetes (SS-D and MS-D, respectively). In BB/W diabetic rats and in rats with SS-D, indices of IAP were significantly increased in tissues and vessels predisposed to diabetic vascular disease in humans, including the eyes (anterior uvea, posterior uvea, and retina), sciatic nerve, aorta, kidney, and new vessels formed after induction of diabetes. No evidence of increased IAP was observed in heart, brain, testes, or skeletal muscle in BB/W or SS-D rats. In MS-D rats, indices of IAP were increased only in the kidney and in new vessels formed after the onset of diabetes. Marked tissue differences were observed in the effects of two structurally different aldose reductase inhibitors (sorbinil and tolrestat) and of castration on diabetes-induced increases in IAP and in tissue levels of polyols in SS-D rats. Both aldose reductase inhibitors and castration completely prevented diabetes-induced increases in IAP in new vessels and in sciatic nerve in BB/W and SS-D rats. Both aldose reductase inhibitors also markedly decreased IAP in the anterior uvea (approximately 85%), posterior uvea (approximately 65-75%), retina (approximately 65-70%), and kidney (approximately 70-100%); castration reduced IAP in the anterior uvea (approximately 55%), kidney (approximately 50%), and retina (approximately 30%) but had no effect on the posterior uvea. The diabetes-induced increases in IAP in the aorta were reduced only slightly (approximately 20%) by aldose reductase inhibitors and castration.(ABSTRACT TRUNCATED AT 250 WORDS)

Permeability changes in blood-retinal barrier of galactosemic rats are prevented by aldose reductase inhibitors.

Permeability-surface-area products (PA) for sucrose at the blood-retinal barrier (BRB) were determined with quantitative in vivo techniques and compared in control rats, rats fed a 50% galactosemic diet, and rats fed a diet containing both galactose and the aldose reductase inhibitor sorbinil. The mean PA +/- SE for controls was 0.656 x 10(-5) +/- 0.13 ml.g-1.s-1 and increased by approximately 500% in galactose-fed animals to 3.13 x 10(-5) +/- 0.32 ml.g-1.s-1. Animals fed both galactose and sorbinil showed no significant difference from control animals (P greater than .05), with a PA of 0.91 x 10(-5) +/- 0.22 ml.g-1.s-1. No breach in the BRB to horseradish peroxidase was detected in any of the groups. These results demonstrate an increased permeability at the BRB to small molecules in galactosemic rats that is prevented by an aldose reductase inhibitor. This suggests that the retinal capillary basement membrane thickening seen in galactosemic rats is associated with an increased permeability of the BRB and that aldose reductase is implicated in its pathogenesis.

Arterial bifurcations in the human retina.

The branching angles and relative diameters of blood vessels in 51 arterial bifurcations in the retina of a normal human eye were measured. In eight other bifurcations, only the total branching angles were measured. The results are compared with theoretical predictions in an attempt to understand the physiological principles governing branching in the cardiovascular system.

Identification and characterization of the insulin receptor of bovine retinal microvessels.

The presence of specific, high affinity receptors for insulin has been demonstrated in purified preparations of bovine retinal microvessels. The binding of [125I]insulin to isolated retinal microvessels was inhibited by unlabeled insulin, but not by other peptide hormones. Scatchard analysis of the [125I]insulin binding data gave a curvilinear plot similar to that exhibited by insulin receptors in known insulin-sensitive tissues such as adipocytes and hepatocytes. Binding of [125I]insulin to retinal microvessels, followed by covalent cross-linking of the bound ligand to the alpha-subunit of the insulin receptor with the bifunctional reagent disuccinimidyl suberate, yielded a prominent specific [125I]insulin-labeled band when analyzed by sodium dodecyl sulfate-gel electrophoresis followed by autoradiography, and this band had a mobility identical to that of the corresponding complex obtained with rat liver plasma membranes (mol wt, 125,000). These results demonstrate for the first time that the retinal microvasculature, a major site of pathological injury in diabetes mellitus, contains insulin receptors that are similar to those present in known insulin-sensitive tissues, such as liver, fat, and muscle.

Cataract formation is prevented by administration of verapamil to diabetic rats.

The purpose of the present study was to examine the effects on cataractogenesis of daily sc administration of the Ca2+ antagonist drug verapamil to diabetic rats. Streptozotocin-induced diabetic rats were given verapamil half-way through the 8-week experimental period or during the full 8 weeks of diabetes. Verapamil administration had no effect on the high blood glucose values, low circulating insulin levels, or elevated triglyceride and cholesterol concentrations in the diabetic rats. Untreated diabetic rats had a 90% incidence of cataracts. Four weeks of verapamil administration reduced this incidence to 41%, and a full 8 weeks of drug treatment further lowered the incidence to 20%. Diltiazem, another Ca2+ antagonist, lowered the incidence of cataracts in the diabetic rats to a similar extent. Verapamil administration to the diabetic animals also partially protected against the presence of retinal microangiopathy in the diabetic animals. Lenticular hydration and lipid accumulation were only indirectly related to cataractogenesis in the diabetic rats and its protection by verapamil treatment. Lenticular electrolyte imbalance, particularly Ca2+, in the diabetic animals was closely correlated with cataract formation, and verapamil significantly reduced the alterations in these ion concentrations. The present results demonstrate the efficacy of verapamil as a protective agent against cataractogenesis and some retinal damage in diabetic animals. Most importantly, this occurs in the absence of any change in the glycemic status of the diabetic animals. The findings strongly support a role for lenticular Ca2+ imbalance in cataract development in diabetes and provide initial evidence to suggest its clinical use in the diabetic population at risk for blindness.

Insulin-like growth factor-I modulates endothelial cell chemotaxis.

Insulin-like growth factor-I (IGF-I) significantly promoted chemotaxis in three separate endothelial cell lines at concentrations ranging from 4 to 150 ng/ml. When added to minimum essential medium (MEM) supplemented with 1% fetal calf serum, IGF-I augmented chemotaxis to a level equalling the maximal chemotactic effect obtainable with MEM supplemented with 10% fetal calf serum (FCS 10%). The data are compatible with a potential role for IGF-I in neovascularization.

Integrity of the blood-brain barrier in retinal xenografts is correlated with the immunological status of the host.

The purpose of this study was to determine the immunological correlates of blood-brain barrier breakdown in retinal xenografts in rats by utilizing skin grafting to initiate a timed immune response to the transplanted neural tissue. Embryonic day 13-14 CD-1 mouse retinae were grafted into the brainstem parenchyma of neonatal Sprague-Dawley rats. In one group of animals a 100 mm2 CD-1 skin graft was placed on the flank 21 days after the initial neural transplant in order to provoke an immune response to the neural graft. Control animals received no skin graft. Animals were injected with horseradish peroxidase (HRP) in the femoral vein 2-8 days after skin grafting. Brains were processed for Nissl, HRP-tetramethylbenzidine, and anti-M-6, -lymphocyte, -macrophage, and -astrocyte antibodies. Experimental and control animals injected 2-4 days after skin grafting showed no leakage of reaction product in the grafted tissue. A small percentage (one of eight) of 5-day animals showed isolated, patchy leakage, but no evidence of rejection of the neural graft. At 6 days all of the grafts showed evidence of leakage, and 71% of these grafts showed infiltration of lymphocytes. By 7-8 days extensive leakage of HRP and widespread infiltration of lymphocytes and macrophages were clearly evident. The present study demonstrates that blood-brain barrier breakdown is correlated closely with the sequence of immunological rejection of the graft. While these results confirm that a barrier exists in healthy neural transplants, they suggest that immunological factors should be considered in cases in which grafts are not protected by an intact barrier.

Relationship between astrocytes, ganglion cells and vasculature of the retina.

We have studied the distribution of astrocytes in the ganglion cell and nerve fibre layers of the retina in cat, rat, rabbit, and possum using anti-serum and a monoclonal antibody against glial fibrillary acidic protein (GFAP) and our own monoclonal antibody against glial filaments. The distribution of retinal astrocytes appears to be strongly determined by the vasculature of the retina; astrocytes are absent from almost all the retina of the possum and from the avascular regions of the rabbit retina. In the cat and rabbit, retinal astrocytes also show a strong affinity for the bundles of ganglion cell axons found at the inner surface of the retina. Retinal astrocytes do not invest the somas of ganglion cells, and even in areas of retina in which they are numerous, they are sharply confined to the layer of ganglion cell axons. It is suggested that retinal astrocytes are "immigrant" fibrous astrocytes that enter the retina with its vasculature.

Cell volume and permeability of oxygen-and glucose-deprived retina in vitro.

Rabbit retina was deprived of O(2) and glucose in vitro for up to four hours at 37 C. Intracellular volume was measured, using inulin as an extracellular marker. After a 30-minute latency, cells swelled rapidly to more than twice normal volume while extracellular volume was unchanged. Intracellular accumulation of water was not reversed by resupply of oxygen and glucose. Permeability to small molecules was assessed with mannitol. The ratio of mannitol space to inulin space averaged 1.0 in controls. This ratio remained 1.0 up to 30 minutes of deprivation, but increased to 1.2 by 60 minutes. Permeability to large molecules was assessed from the rate of loss of isotopically labeled cell protein into the medium. There was no difference between control and deprived retinas up to three hours.

Further observations on cerebral or retinal ischemia in patients with right-left intracardiac shunts.

Between October 1983 and November 1986, 20 patients suspected of having a paradoxical cerebral or retinal embolism were identified. Cerebral infarction was the most common presentation. Six patients had a patent foramen ovale demonstrated by cardiac catheterization or surgery. Three patients had a newly discovered atrial septal defect, and one had an atrial septal defect that had previously been treated surgically. Results of the cardiac physical examination, chest roentgenography, and electrocardiography were unremarkable in 12 patients. Eighteen patients had a right-to-left shunt demonstrated by contrast echocardiography. Nine of these patients underwent cardiac catheterization; seven had abnormal catheterization study results: four had a patent foramen ovale and three had an atrial septal defect. Sixteen patients received medical therapy only while four underwent surgery. All patients survived the initial insult. There were no deaths or ischemic recurrences on follow-up ranging from one month to three years. We believe that contrast echocardiography can provide important diagnostic information, even in situations in which cardiac involvement is not suspected. Additional studies are needed before the optimal treatment of presumptive paradoxical cerebral embolism can be determined.