The nature of the process leading to the acellular nonperfused capillaries of diabetic microangiopathy remains unknown. Because these capillaries manifest thickened basement membranes, we asked whether the process causing deposition of excess extracellular matrix in diabetes modifies cell-matrix interactions in a direction that would compromise cell renewal. In 44 individual isolates of human umbilical vein endothelial cells we observed that high glucose concentrations (30 mM) induce coordinate increases in the levels of mRNAs encoding fibronectin and the fibronectinspecific integrin receptor as5fl as well as in the cognate proteins. Expression of the integrin subunit a3, component of the a3f81 polyspecific receptor for fibronectin, laminin, and collagen, was also up-regulated by high glucose. Overexpression of integrins correlated with increased cell attachment to exogenous fibronectin and laminin as well as to complex matrix. Moreover, cells exhibited firmer steady-state adhesion to their own matrix. To correlate these in vitro observations with events in human diabetic retinopathy we measured integrin levels in retinal trypsin digests prepared from 10 patients with 8.2 ± 1.6 (mean ± SE) years of diabetes and 10 age-and sex-matched nondiabetic controls. Microvessels of diabetic patients showed increased immunostaining for B13 integrin (P = 0.025) when compared with control microvessels. These data show that high glucose and diabetes increase integrin expression and thus alter the interaction of vascular endothelial cells with their basement membranes in the direction of firmer cell-matrix adhesion. This could compromise the migration and replication critical to the reendothelialization process and contribute to microvascular occlusion.The most characteristic chronic sequelae of diabetes mellitus (retinopathy leading to blindness, nephropathy leading to renal failure) are local manifestations of a widespread occlusive microangiopathy (1-3). Histologically the nonperfused capillaries are acellular and surrounded by thickened basement membranes, which represent the identifying feature of diabetic microangiopathy (4). The nature of the process leading to accelerated cellular demise and whether it relates to the accumulation of basement membrane material have remained outstanding issues (5).Among the modulatory influences exerted by the extracellular matrix on endothelial cells, some are specifically related to the amount of matrix. It has been shown that increasing concentrations of fibronectin and, to a lesser extent, of laminin and collagen IV reduce endothelial cell migration and replication (6). Because these functions are critical to the reendothelialization process (6), it could be hypothesized that diabetic vessels become occluded because excess matrixThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. compromises prompt and efficient reendothe...

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We have investigated whether antisense oligonucleotides delivered intravitreally could reduce gene expression specifically in the retina. In this study, phosphorothioate antisense oligonucleotides targeted to fibronectin transcripts were coupled to a novel carrier and used to specifically reduce fibronectin (FN) expression in retinal vascular cells. Using confocal microscopy, fluorescence from fluorescein isothio-cyanate-labeled FN-oligonucleotides was detected in retinal vascular cells at 24 h postinjection and persisted until day 6 (the end point of this study). The fibronectin mRNA level was consistently decreased to 86.7% +/- 7.9% of control (p<0.05) at day 2, and 46.7% +/- 4.9% of control (p<0.01) at day 6. In contrast, the beta-actin mRNA level, an internal control, was unaltered in rat retinas that received FN-oligonucleotides. Fibronectin protein level at day 6 was also significantly reduced to 61.4% +/- 16% of control (p<0.01). No toxic effect resulting from the carrier was detected histologically. Thus, intravitreal delivery of antisense oligonucleotides to modulate abnormal gene expression in retinal diseases may be an effective approach for ocular gene therapy.

Most studies addressing how cellular phenotypes are affected by the extracellular matrix (ECM) have contrasted cellular responses to different ECM molecules or combinations of molecules, and provided paradigms for interpreting histogenesis, differentiated function, and tissue repair [1][2][3]. We are interested in the possibility that learning whether and how cellular phenotypes are also affected by quantitative changes in the underlying or surrounding ECM may provide paradigms for interpreting pathologies characterized by excess ECM accumulation. One such pathology is diabetic microangiopathy. The process, initiated by the metabolic abnormalities of diabetes, is hallmarked by increased thickness of vascular basement membranes and excess ECM accumulation in the renal mesangium, and leads eventually to microvascular obliteration and the well known clinical sequelae of diabetes [4]. Mesangial expansion is thought to play a pathogenetic role in the loss of glomerular Diabetologia (1997) 40: 879-886 Cytoskeletal changes induced by excess extracellular matrix impair endothelial cell replication m g/ml) of exogenous fibronectin, when compared with HUVEC plated on tissue culture plastic or 0.25, 1.0, and 5.0 m g/ml fibronectin; the decreased replication was attributable to delayed transit through the G 1 phase of the cell cycle. HU-VEC grown on both 1 and 10 m g/ml fibronectin exhibited a modest upregulation of the fibronectinspecific integrin receptor a 5b1, and increased attachment to fibronectin substratum. However, unique to the HUVEC plated on growth-inhibitory concentrations of fibronectin was a redistribution in situ of integrins and vinculin to form more numerous focal adhesions, and an increased polymerization of cytoskeletal actin to form stress fibers. Concentrations (0.01 m g/ ml) of cytochalasin D intended to prevent excess actin polymerization prevented the growth inhibition. Thus, excess ECM hampers endothelial cell replication in vitro through increased cell-ECM adhesion and attendant cytoskeletal rearrangements. These phenotypic changes provide probes to test whether cell-ECM interactions are altered in diabetic vessels in a direction that may compromise orderly endothelial cell renewal and its antithrombogenic function. [Diabetologia (1997)