Studies on retinal capillary cells in tissue culture.
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Biomedical subjects
Publications and source records attributed to R N Frank.
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Patients with sickle cell anemia may be zinc-deficient. Zinc deficiency has been reported to cause delayed dark adaptation. We have studied dark adaptation in 13 patients with homozygous sickle cell anemia. Six patients had delayed dark adaptation. Their mean plasma and neutrophil zinc levels were 93.83 +/- 8.26 micrograms/100 microliters and 37.33 +/- 11.88 micrograms/10(10) cells, respectively, compared to the corresponding mean values for seven patients with normal dark adaptation of 105.86 +/- 13.31 and 86.71 +/- 19.53. For the six patients with delayed dark adaptation, the lower neutrophil zinc value, but not the plasma value, was significant (p less than 0.01). Three of the six were treated with oral zinc, with improvement in their dark adaptation. Neutrophil zinc levels correlated inversely (r = -0.77, p less than 0.01) with log relative threshold at 30 min. We conclude that patients with sickle cell anemia may show decreased ability to dark-adapt that is related to zinc deficiency.
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To study objectively the epidemiology of retinopathy in juvenile-onset diabetes, we performed fundus photography and fluorescein angiography, using the Diabetic Retinopathy Study protocol, on 122 juvenile diabetics and 65 demographically similar non-diabetic subjects as the control group. Photographs and angiograms were masked as to subjects' identities and evaluated independently by five retinal subspecialists. There was no retinopathy in control subjects. In diabetics, prevalence of retinopathy increased with the duration of disease, being 0% after zero to four years, 27% for five to nine years, and 71% for more than ten years. Retinopathy also increased in prevalence with age with a sharp rise after age 15. There is indication that age and duration act independently. Details of our method for establishing the diagnosis of diabetic retinopathy are presented, together with the degree of observer variability in identifying early lesions.
The cells that proliferate from microvessels isolated from calf retinas and placed in tissue culture have been found to derive from the intramural pericytes. When these cells were cultured in medium supplemented with ascorbate and pulsed for 24 hr with [14C]proline, about 2% of the [14C]proline in the nondialyzable protein secreted into the culture medium by these cells was hydroxylated, and about 10% of the incorporated [4C]proline was solubilized by purified collagenase. Agarose gel chromatography of the postculture media showed that unreduced [14C]collagen polypeptides were recovered in large-molecular-weight aggregates (greater than 300,000 daltons) which were largely converted to chains of approximately 95,000 molecular weight and some dimers and trimers of MW approximately 180,000 and 270,000 when the medium was subjected to reduction and alkylation prior to chromatography. The findings indicate that cultured retinal pericytes elaborate collagen and suggest the production of type III collagen. Retinal microvessel cells in culture may facilitate study of abnormalities in retinal pericyte function and collagen synthesis that occur in retinal vascular diseases.
A 33-year-old black woman with hemoglobin SC disease and a history of photocoagulation for peripherally located retinal neovascularization had a neovascular frond at the temporal border of her right macula. Multiple zones of black sunburst hyperpigmentation were located nearby. Fluorescein angiography showed that the lesions were closely related to an extensive zone of avascular retina in the posterior pole. The perifoveal vasculature was uninvolved, and the patient was unaware of any visual deficit. Although sickle cell maculopathy is a well-recognized entity, there have been no published reports, to the best of our knowledge, of posterior pole neovascularization in patients with sickle cell hemoglobinopathy. The fluorescein angiographic findings in our case provide further support for the hypothesis that retinal hypoxia is an important stimulus for retinal neovascularization.
When microvessel (predominantly capillary) fragments freshly isolated from the retinas of young kittens are incubated in tissue culture medium, we observe the slow outgrowth of cells that appear to be derived from the capillary endothelium. The cells grow as a monolayer with a tightly packed, mosaic-like array. Electron microscopy of these cells reveals "tight junctions" (zonulae occludentes) resembling those found in the endothelium of intact capillaries. The availability of in vitro preparations of retinal capillary endothelial cells should facilitate investigations of the causes of abnormal endothelial cell proliferation in various diseases and of factors that modify cell junctions and capillary permeability in the retina. Since we have previously demonstrated proliferation in culture of intramural pericytes ("mural cells") from retinal capillaries, it should now be possible to carry out compratative studies of biochemical and functional properties of retinal capillary pericytes and endothelial cells.
We studied 29 diabetic patients (eight without and 21 with retinopathy) and 29 matched control subjects for hemoglobins Ala-c and plasma levels of fibrinogen, von Willebrand factor, and factor VIII/von Willebrand factor antigen. Hemoglobins Ala-c were elevated in all diabetic patients, regardless of retinopathy (P less than 0.001); fibrinogen was elevated only in those with retinopathy (P less than 0.001); and plasma von Willebrand factor was clearly elevated in those with proliferative retinopathy and minimally, if at all, elevated in those with background or no retinopathy. Plasma from five diabetic patients with proliferative retinopathy and five control subjects was tested for its ability to enhance the ADP-induced platelet aggregation of normal or von Willebrand platelet-rich plasma; no differences were found. We conclude that elevated levels of fibrinogen and von Willebrand factor, both recognized plasma cofactors of platelet function, are associated with proliferative diabetic retinopathy. A plasma factor that enhances ADP-induced platelet aggregation could not be found in diabetic plasma, and we doubt that increased von Willebrand factor is responsible for its finding by others.
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Cultured mural cells (intramural pericytes) from adult rhesus retinal capillaries were examined for the presence of the sorbitol pathway. A radioimmunoassay for human aldose reductase, cross-reactive with rhesus lens aldose reductase, showed the presence of this enzyme in our cultured cells. Mural cells grown in culture media containing normal (10 mM) and high (40 mM) levels of glucose were examined for polyol accumulation by gas-liquid chromatography. Cells incubated in high glucose medium showed a threefold increase in sorbitol concentration over cells grown at low glucose levels. After 30 days in high glucose medium, mural cells formed dense multilayered areas with extensive cellular debris. These findings suggest the presence of the sorbitol pathway in cultured retinal mural cells and cellular degeneration in high glucose medium; this may have possible implications in the pathogenesis of diabetic retinopathy.
Capillaries from bovine, monkey, and human retinas maintained in tissue culture produced a monolayer of cells. Autoradiographic and electron microscopic evidence indicated that the mural cells (intramural pericytes) were the cells that proliferated. Since intramural pericytes are damaged selectively in diabetes mellitus, their availability in culture will be useful in seeking means to control diabetic retinopathy.
Partial separation of protein kinase activity from rhodopsin in isolated bovine retinal photoreceptor outer segments was accomplished by mild ultrasonic treatment followed by ultracentrifugation. Residual kinase activity in the rhodopsin-rich sediment was destroyed by chemical denaturation which did not affect the spectral properties of the rhodopsin. The retinal outer segment kinase was found to be specific for rhodopsin, since in these preparations it alone of several bovine protein kinases was capable of phosphorylating rhodopsin in the light. The phosphorylation reaction apparently requires a specific conformation of the rhodopsin molecule since it is abolished by heat denaturation of rhodopsin, and it is greatly reduced or abolished by treatment of the visual pigment protein with potassium alum after the rhodopsin has been "bleached" by light. When kinase and rhodopsin or opsin fractions were prepared from dark-adapted and bleached outer segments and the resultant fractions were mixed in various combinations of bleached and unbleached preparations, the observed pattern of light-activated phosphorylation was consistent only with the interpretation that a conformational change in the rhodopsin molecule in the light exposes a site on the visual pigment protein to the kinase and ATP. These results rule out the possibility of a direct or indirect (rhodopsin-mediated) light activation of the kinase. Finally, phosphorylation of retinal outer segment protein in monochromatic lights of various wavelengths followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that both rhodopsin and the higher molecular weight visual pigment protein reported by several laboratories have the same action spectrum for phosphorylation. This result is consistent with the suggestion that the higher molecular weight species is a rhodopsin dimer.
Twenty-four patients with proliferative or preproliferative diabetic retinopathy underwent extensive argon laser photocoagulation in one eye. Detailed central and peripheral visual-field examinations and electroretinography were performed before treatment, and at intervals after treatment. Visual field changes were as follows: (1) eight patients had only mild to moderate constriction of all isopters, (2) 11 had discrete scotomata in addition, (3) two had prominent nerve-fiber-bundle defects, (4) three had severe constriction of all isopters, save (in two) that to the largest peripheral test object. Electroretinographic b-wave amplitudes were reduced an average of 40% to white test flashes. Blue test flashes showed an even larger reduction of the rod response. This suggests the receptors in approximately 40% of the retinal area are destroyed by such extensive photocoagulation, covering predominantly that part of the retina that has the highest concentration of rods.
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