Parameters of genome instability and morphological alterations associated with cell transformation were studied in an isogeneic set of clonal human uroepithelial cell (HUC) lines immortalized by the human papilloma virus 16 (HPV16) E6 and/or E7 gene(s). HPV16 E6 binds p53, leading to rapid degradation of p53, whereas E7 binds and alters pRb and other proteins. We report that two independent E7-immortalized HUC lines showed minimal phenotypic or genotypic alterations, except that both lines contained amplification of 20q DNA sequences and a greater polyploidization at an early passage. The E7-immortalized HUC line resembled normal HUC lines, except that they failed to senesce. In contrast, the E6-immortalized HUC lines were morphologically altered, contained numerous random chromosome aberrations, and showed unstable evolving karyotypes with passage in culture. No amplified DNA sequences were detected in E6-immortalized HUC lines. Instead, clonal losses of chromosome regions (i.e., -3p, -6q, -9p), putatively containing tumor suppressor or senescence genes, accompanied the E6-HUC immortalization event. E6-immortalized HUC lines showed transformed phenotypes similar to E6/E7-HUC lines. The difference in genome stability between E6- and E7-immortalized HUC was highly significant statistically (p-value < 10(-6). Thus, the HPV16 E7 gene led to HUC immortalization by a pathway that blocked cellular senescence, but did not disrupt genome stability. These results implicate p53 loss, but not pRb alteration, in genome destabilization.

PURPOSEThe purpose of this study was to examine retinal capillaries and their pericytes that previous research suggests to be contractile. A contractile role regulating capillary blood flow may be more apparent when the vasculature is subjected to the stress of systemic hypertension.METHODSUsing ultrastructural morphometry and the myosin subfragment-1 technique, retinal capillaries of normal and hypertensive rats were measured at three different time points, early, intermediate, and late (24, 44, and 68 wk).RESULTSHypertensive capillaries seemed to dilate at the early time point (P = 0.002), were constricted at the intermediate time point (P < 0.001), and did not redilate later. Wall thickness was enlarged at all times, pericyte coverage (the ratio of plasma membrane length in contact with the vascular circumference to the outer circumference of the endothelial tube) was greater at early and intermediate time points, and the total area of viable cytoplasm relative to the vessel wall area was increased at the intermediate time (all P < 0.001). Also, at the intermediate time, the circumferential coverage of the endothelial tube by actin filament bundles within pericytes and the actin area relative to the vessel wall area had increased (P < 0.001).CONCLUSIONSThese data indicate that the effects of systemic hypertension extend into the retinal capillary bed, causing pericyte change with actin increase and capillary constriction. They represent the first in vivo indirect evidence by morphologic criteria for pericyte contractility in retinal vascular disease.

Macular edema and collateral vessels were examined clinically and histopathologically up to 48 months after branch retinal vein occlusion in six eyes of five cynomolgus monkeys. In all six, central macular swelling and fluorescein leakage from the retinal vasculature were confined to the acute stage. However, histopathologically, at the chronic stage, only two maculas were completely recovered and unremarkable, whereas the other four showed variable degrees of cystoid degeneration and photoreceptor cell loss. In the two recovered maculas, six to eight normal-sized capillaries separated the fovea from the nearest cluster of capillary collaterals. In three maculas with cystic degeneration, collaterals incorporated the circumfoveal capillaries. In the fourth macula with cystic degeneration, collaterals were separated from the center by two normal-sized capillaries but were also associated with large areas of capillary nonperfusion partially due to occlusion of the macular arteriole.

Previous studies of fibronectin (FN) distribution in eye tissue have relied on immunofluorescence (IF) techniques on frozen sections, and have not included the rat. Using rat eyes, a technique was developed for immunoperoxidase (IP) staining of formalin fixed paraffin embedded material, and the results were compared to those obtained by IF. IP was technically more difficult, and required pepsinization of tissue after formalin fixation to obtain consistent results. The optimum pepsin time varied for different structures in the eye. IP offers better tissue preservation and stain resolution. Results with IF were consistent with those observed with the newly applied IP. The distribution of FN in rat eyes was similar though not identical to that reported in other species. Prominent stain was observed in the conjunctiva, basement membrane of the corneal epithelium, corneal stroma, anterior aspect of Descemet's membrane, trabecular meshwork, perivascular stroma of the ciliary body, choroid and retinal blood vessels. Lens structures, vitreous, and internal limiting membrane of the retina were negative.

A patient with proliferative diabetic retinopathy was treated by panretinal and focal photocoagulation. Later, he developed one area of clinically diagnosed chorioretinal and choriovitreal neovascularization (CNV), neovascular glaucoma, and a blind painful eye necessitating enucleation. Clinicopathologic correlations of this eye including fundus photography, fluorescein angiography, light and electron microscopy are reported. Histopathologic examination revealed three areas of CNV, suggesting that some CNV may go undetected clinically also in other cases and thus may occur more frequently than evident from the literature. Our CNV occurred at sites of focal treatment. Retreatment of one area was unsuccessful. Choriovitreal neovascularization passed through discontinuities of Bruch's membrane into the retina and showed fenestrae of the endothelial cells. Endothelial fenestrae may account for the profuse fluorescein leakage seen clinically in CNV.

The eye of a patient with age-related macular degeneration was treated with krypton laser photocoagulation and later studied histopathologically. Five months after treatment the original membrane was completely obliterated, but a separate new membrane was found, which was fed by the retinal circulation and not by the choroid. The frequency of such recurrences is not known, but they may be related to krypton laser treatment.

In 14 patients, preretinal membranes, causing retinal traction and severe visual impairment, were removed by vitrectomy and evaluated by light and electron microscopy using myosin subfragment-1 to stain actin filaments. Eight membranes were of vascular origin, six of nonvascular origin. All but one contained bundles of oriented actin filaments within a number of their nonvascular stroma cells, suggesting that the contractile protein action may have been involved in their clinically observed contraction.