Peter A. Campochiaro

Retinal pigment epithelial cells produce PDGF-like proteins and secrete them into their media☆

Human retinal pigment epithelial cells at confluence were used to condition serum-free Dulbeccos modified Eagles medium. Conditioned media were exhaustively dialyzed against 0.5 N acetic acid, lyophilized, and subjected to Western blot analysis, using as primary antibody an IgG fraction prepared from goat antiserum directed against human platelet-derived growth factor. Native platelet-derived growth factor was resolved as a band with Mr of 30 kDa under non-reducing conditions, while bands with Mr of 36-38 kDa and 18.5 kDa were resolved from retinal pigment epithelial cell-conditioned media. Acid extracts of retinal pigment epithelial cells also contained bands at 36-38 kDa and media conditioned for 48 hr exhibited much denser bands than media conditioned for 24 hr. No bands were detected when non-immune goat IgG fractions were substituted for primary antibody and when conditioned media were prepared from several human fibroblast lines in the same manner as those prepared from retinal pigment epithelial cells, no detectable bands or only a faint shadow at 36 kDa were seen. Retinal pigment epithelial cell-conditioned media prepared in the presence of [35S]methionine were loaded on an anti-platelet-derived growth factor IgG affinity column, eluted, and subjected to SDS-polyacrylamide gel electrophoresis. Bands with Mr slightly less than 36 kDa and 18 kDa were visualized by autoradiography, demonstrating that the platelet-derived growth factor-like proteins in retinal pigment epithelial cell-conditioned media are newly synthesized. Two fractions eluted from the column also markedly stimulated fibroblast chemotaxis and incorporation of [3H]thymidine, both of which were neutralized by soluble anti-platelet-derived growth factor IgG. These data suggest that retinal pigment epithelial cells in culture produce platelet-derived growth factor-like proteins and secrete them into their media where they are capable of stimulating fibroblast chemotaxis and proliferation.

Retinal Pigment Epithelial Cells Release inhibitors of Neovascularization

Human retinal pigment epithelial (RPE) cells in culture were found to release a substance (or substances) that causes the regression of new blood vessels on the chick embryonic yolk sac and inhibits proliferation of fetal bovine aortic endothelial cells and human retinal microvessel endothelial cells in vitro. Neither astrocytes nor fibroblasts under identical test conditions released detectable inhibitors of neovascularization or endothelial cell growth. Subconfluent and superconfluent cultures of human RPE cells released higher levels of inhibitor than confluent cultures.

Aldose Reductase Expression in Human Diabetic Retina and Retinal Pigment Epithelium

The conversion of glucose to sorbitol by aldose reductase (AR) and its subsequent intracellular accumulation have been implicated in the pathogenesis of diabetic cataracts. There is also evidence linking AR activity with retinal capillary basement membrane thickening in galactosemic rats, suggesting a possible role in diabetic retinopathy. In this study, we explored one feature of this issue by examining diabetic and nondiabetic eyes for immunoreactive AR. AR was immunohistochemically undetectable in the retinal pigment epithelia (RPE) and neural retinas of nondiabetic human eyes. Weak, focal staining for AR was present unilaterally in the RPE of 1 of 11 diabetic patients without pathologic ocular findings and in 43% of diabetic patients with mild ocular findings. Retinal positivity was found (unilaterally) in only 2 of 19 individuals from either of these mildly affected groups. Fifty-five percent of patients with background retinopathy demonstrated AR positivity in the RPE, and half of these expressed AR in the RPE of both eyes. Of the individuals with proliferative diabetic retinopathy, 87.5% showed bilateral staining in the RPE. Retinal positivity was present in 36% of background retinopathy and 75% of proliferative retinopathy cases, demonstrating a positive correlation between AR expression and the severity of the disorder. In weakly staining retinas, only the ganglion cell bodies, nerve fibers, and Müller cells were positive, whereas in intensely staining cases, virtually the entire retina, except for the rod outer segments, was positive. Eyes from patients who had had diabetes ≤6 yr were negative for AR, but those from long-term-diabetic patients (14–45 yr)manifested positivity. These data suggest that enhanced expression of AR may play a role in the development of diabetic retinopathy.

Ultrastructural localization of blood-retinal barrier breakdown in diabetic and galactosemic rats.

Breakdown of the blood-retinal barrier (BRB) is an early event in diabetic and galactosemic rats, but the location and nature of the specific defect(s) are controversial. Using an electron microscopic immunocytochemical technique, the retinas of normal, diabetic, and galactosemic rats were immunostained for endogenous albumin. Normal rats showed little evidence of BRB breakdown at either the inner barrier (retinal vasculature) or the outer barrier (retinal pigment epithelium) (RPE). In diabetic and galactosemic rats, as was true in human diabetics, BRB breakdown occurred predominantly at the inner BRB, but in some cases at the outer barrier as well. Treatment with the aldose reductase inhibitor sorbinil largely prevented BRB failure in galactosemic rats. In the inner retina of diabetic and galactosemic rats, albumin was frequently demonstrated on the abluminal side of the retinal capillary endothelium (RCE) in intercellular spaces, basal laminae, pericytes, ganglion cells, astrocytes, and the perinuclear cytoplasm of cells in the inner nuclear layer. Albumin did not appear to cross RCE cell junctions; however, it was occasionally seen in RCE cytoplasm of galactosemic rats. In the outer retina, albumin was frequently detected in the subretinal space, in the intercellular space between photoreceptors, and in the perinuclear cytoplasm of photoreceptor cells, but was only infrequently found in the RPE cells constituting the barrier. Albumin derived from the choroidal vasculature did not appear to cross the tight junctions of the RPE. These findings suggest that specific sites of BRB compromise are infrequent but that once albumin has crossed the RCE or RPE it freely permeates the retinal tissue by filling intercellular spaces and permeating the membranes of cells not implicated in BRB formation. The diffuse cytoplasmic staining of some RCE and RPE cells suggests that the predominant means of BRB breakdown in diabetes and galactosemia involves increased focal permeability of the surface membranes of the RCE and RPE cells rather than defective tight junctions or vesicular transport.

Human retinal pigment epithelial cells possess muscarinic receptors coupled to calcium mobilization

Human retinal pigment epithelial (RPE) cells in culture demonstrated saturable specific binding of [3H]quinuclidinyl benzilate (QNB). Specific binding represents about 75% of total binding. Scatchard analysis yields a Kd of 0.178 nM and Bmax of 42 fmol/mg protein. Atropine and carbachol show typical displacement curves, and a Hill plot has a slope of 0.96, suggesting a homogeneous population of receptors. Muscarinic agonists have no effect on intracellular cyclic adenosine monophosphate levels in RPE cells measured by radioimmunoassay, nor do they alter the isoproterenol-induced stimulation of adenylate cyclase. However, both acetylcholine and carbachol cause a rapid increase in intracellular calcium concentration measured by the fluorescent indicator quin 2. Atropine reverses the calcium rise when added after agonist and prevents the rise when added prior to agonist. These data suggest that human RPE cells possess muscarinic receptors coupled to calcium mobilization.

Spontaneous Involution of Subfoveal Neovascularization

We studied five patients who maintained or spontaneously regained significant central vision despite the presence of a subfoveal neovascular membrane. Sequential photographs and fluorescein angiograms showed a pattern of development common to these patients and not observed in patients who lose all central vision. The pattern involves formation of a pigmented ring around the subfoveal neovascular membrane followed by alteration of the membrane from one that leaks fluorescein to one that stains but does not leak. This pattern is associated with gradual resorption of subretinal fluid and apparent limitation of subretinal hemorrhage and fibrosis. This course suggests the occurrence of spontaneous involution of some subfoveal neovascular membranes and implicates the retinal pigment epithelium in this process.

Characterization of adenylate cyclase in human retinal pigment epithelial cells in vitro.

Human retinal pigment epithelial cells in culture demonstrate adenylate cyclase activity. It is membrane-bound and modulated by GTP regulatory proteins. It is effectively activated only by beta-adrenergic agonists (L-isoproterenol greater than or equal to L-epinephrine greater than L-norepinephrine) and some prostaglandins (PGE1 and PGE2, but not PGF1 alpha). The adrenergic response appears to be mediated by beta-2 receptors. No inhibitory ligands could be demonstrated. Its characteristics, which are similar to functional adenylate cyclase complexes in other mammalian cells, and its selective and sensitive agonist responsiveness, suggest a possible physiologic role in the regulation of human retinal pigment epithelial-cell function.

Human retinal pigment epithelial cells in culture possess A2-adenosine receptors

Adenosine agonists cause a marked stimulation in cyclic AMP accumulation in whole human retinal pigment epithelial (RPE) cells in the presence of adenosine deaminase and papaverine, a phosphodiesterase inhibitor. N-Ethylcarboxamidoadenosine (NECA) stimulates cyclic AMP accumulation 16.1-fold above basal with an EC50 of 2.5 x 10(-7) M. It is also an effective (1.9-fold) stimulator of adenylate cyclase activity in RPE membrane preparations and a modest (1.22-fold) stimulator in the presence of forskolin in RPE cell membranes prepared from freshly isolated porcine RPE. N6-Cyclopentyladenosine (CPA) and N6-phenylisopropyladenosine (PIA) also increase cyclic AMP levels with EC50s of 4.9 x 10(6) M (8.9-fold above basal) and 3.5 x 10(-6) M (8.0-fold above basal) respectively. This potency order (NECA greater than PIA greater than CPA) is typical of A2-adenosine receptors. The relatively A1-selective agonists 10(-7) M indicating that RPE cells do not have A1-receptors which inhibit adenylate cyclase. Three adenosine receptor antagonists, BW-A1433U, 8-cyclopentyltheophylline and 8-sulfophenyltheophylline, blocked the NECA-induced stimulation of cyclic AMP accumulation with IC50s of 0.36 microM, 1.5 microM, and 75 microM respectively. Since alteration of cAMP levels has been demonstrated to affect several RPE functions, including cell migration, resorption of subretinal fluid, and phagocytosis, adenosine may play a significant regulatory role in RPE.

Retinal vascular occlusion without retrobulbar or optic nerve sheath hemorrhage after retrobulbar injection of lidocaine

We report a case of retinal vascular occlusion in a patient with severe diabetic retinopathy after retrobulbar injection of lidocaine. Several features of the occlusion are of interest: 1) rapid onset and relatively rapid reversal temporally associated with intervention; 2) numerous areas of focal vascular constriction; 3) absence of retrobulbar hemorrhage or dilated optic nerve sheath on CT scan; and 4) recurrence of nonperfusion after a second injection into the inferior peribulbar space. This suggests that patients with severe vascular disease may suffer retinal vascular occlusion after retrobulbar injections in the absence of identifiable retrobulbar or intraoptic nerve sheath hemorrhage. Though the mechanism is uncertain, this unusual complication deserves consideration, since its early recognition could possibly be of benefit in the management of some patients.

Progressive ultrastructural damage and thickening of the basement membrane of the retinal pigment epithelium in spontaneously diabetic BB rats.

Ultrastructural examination of the neural retina and retinal pigment epithelium was performed in spontaneously diabetic BB rats and non-diabetic littermate controls at various time points during the course of diabetes. Four types of progressive changes occur in diabetic rats: (1) There is a progressive thickening of the basal laminae of the retinal pigment epithelium and the retinal capillaries. (2) In addition to this thickening, there is a progressive, often focal, thickening of the rest of Bruchs membrane, and by 11 1/2 months it contains numerous randomly oriented collagen fibrils. (3) There are progressive ultrastructural changes in retinal pigment epithelial cells consisting of marked dilation of the basal infoldings, the formation of large vacuoles and cell swelling. (4) Rare degenerative changes were noted in retinal capillary pericytes. Since non-diabetic littermates do not show comparable changes, it is likely that they are diabetes-related, suggesting that progressive damage to the retinal pigment epithelium may occur during the course of diabetes.