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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 1, 2004 as doi:10.1096/fj.03-1476fje. |
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* Department of Vitreoretinal Surgery and Angiogenesis Laboratory, Center of Ophthalmology,
Center for Molecular Medicine (ZMMK) University of Cologne, Köln, Germany;
Retina Research Laboratory, Massachusetts Eye and Ear Infirmary,
Laboratory for Surgical Research, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA;
|| Departments of Medicine and Ophthalmology and Center for Diabetes Research, Case Western Reserve University, Cleveland, Ohio, USA; and
Eyetech Research Center, Woburn, Massachusetts, USA
1Correspondence: A.M.J., Department of Vitreoretinal Surgery, Center for Ophthalmology, University of Cologne, Joseph Stelzmannstr. 9, 50924 Köln, Germany; E-mail: JoussenA{at}aol.com; A.P.A., Eyetech Research Center, 42 Cummings Park, Woburn, MA 01801, USA; E-mail: tony.adamis{at}eyetk.com
SPECIFIC AIMS
Diabetic retinopathy, the leading cause of blindness, is characterized by early retinal microvascular dysfunction. As we have shown before, endothelial damage is causally linked to increased leukocyte adhesion and leads to blood-retinal barrier breakdown and diabetic macular edema, the main cause of vision loss in diabetes. Earlier work has established the role of CD18/ICAM-1-mediated leukocyte adhesion in the pathogenesis of early diabetes-induced leukostasis and blood-retinal barrier breakdown. Later on in the course of diabetes, areas of angiographic nonperfusion in vivo frequently colocalize to regions full of acellular capillaries (i.e., basement membrane tubes devoid any viable endothelial cells or pericytes). These acellular capillaries are a hallmark of advanced diabetic retinopathy in humans and in animal models of diabetes. The effect of chronic CD18- and ICAM-1-mediated leukocyte adhesion on the long-term functional and anatomic pathology of diabetic retinopathy remains unknown. To address this issue, we studied the incidence of the signature pathologies of diabetic retinopathy (leukostasis, vascular leakage, and histologic changes) in mice deficient in ICAM-1 and CD18 genes in two established models of chronic diabetic retinopathy
PRINCIPAL FINDINGS
1. CD18 and ICAM knockout mice exhibit decreased leukostasis in two models of long-term diabetes
Adherent leukocytes were quantified in the retinal vasculature after in situ labeling with FITC-concanavalin A. After 11 months of diabetes, the number of adherent leukocytes in wild-type (C57BL/6J) mice was 3.7-fold greater than in age-matched nondiabetic wild-type controls (P<0.001). In contrast, the number of adherent leukocytes in the diabetic CD18–/– or ICAM-1–/– (C57BL/6J) mice did not differ significantly from that of nondiabetic wild-type controls or nondiabetic CD18–/– and ICAM-1–/– age-matched controls (P>0.05 for all).
2. CD18 and ICAM knockout mice bear decreased numbers of injured endothelial cells in two models of long-term diabetes
Endothelial cell injury was investigated with propidium iodide, a fluorophore excluded from uninjured cells but whose uptake is diagnostic for injured and/or dying cells. Little or no endothelial injury was observed in retinas of nondiabetic wild-type, ICAM-1–/–, or CD18–/– mice, but a marked increase in endothelial cell injury was detected in the retinal vessels of 11-month diabetic wild-type mice (P<0.001). Diabetic CD18–/– and ICAM-1–/– mice had markedly less endothelial cell injury (P<0.001 vs. age-matched wild-type diabetic mice).
3. CD18 and ICAM knockout mice exhibit decreased blood-retinal barrier breakdown in two models of long-term diabetes
Permeation of the tracer Evans blue into the retina was used as a parameter of blood-retinal barrier breakdown and evaluated. Evans blue can be used to quantitatively assess subtle degrees of blood-retinal barrier breakdown. After 11 months of diabetes, blood-retinal barrier breakdown was markedly increased in wild-type diabetic animals (P<0.0005 vs. age-matched nondiabetic wild-type controls). In contrast, diabetic CD18–/– and ICAM-1–/– mice manifested 90% and 87% less blood-retinal barrier breakdown, respectively, than wild-type age-matched diabetic mice (P<0.001).
4. CD18 and ICAM knockout mice show less severe histopathological changes in two models of long-term diabetes
Retinal vascular lesions were assessed via trypsin digestion, a method that permits detailed analysis of the retinal vascular anatomy in isolation. After 11 months of diabetes, retinas of the wild-type C57/B16 mice possessed 3.8-fold more acellular capillaries (P<0.001), a pathology that was suppressed by 60% and 56% in diabetic CD18–/– and ICAM-1–/– mice, respectively (P<0.0001 vs. age-matched diabetic wild-type mice). In nondiabetic animals, ICAM-1 or CD18 deficiency did not alter the number of acellular capillaries, which remained low as the animals aged (P>0.05 vs. age-matched wild-type controls).
Endothelial cells form the monolayers that line the intralumenal surface of viable capillaries. In 11-month wild-type diabetic mice, the number of endothelial cells was reduced by 29% (P<0.005 vs. age-matched nondiabetic wild-type controls) whereas endothelial cell counts in the ICAM-1–/– and CD18–/– mice were normal (P>0.05 vs. nondiabetic wild-type mice).
The appearance of pericyte "ghosts" (dead pericytes) is a signature lesion of diabetic retinopathy. In CD18- and ICAM-1-deficient mice, diabetes-induced increases in pericyte death were suppressed by 55% and 66%, respectively (P<0.0001 vs. age-matched 11 month-diabetic wild-type mice). In agreement, the number of normal-appearing pericytes in the diabetic ICAM-1–/– and CD18–/– mice was significantly greater than in age-matched diabetic wild-type controls (P<0.05) and did not differ significantly from that of age-matched nondiabetic wild-type controls.
ICAM-1 or CD18 deficiency inhibited the development of more advanced retinopathy in the galactosemic mice. These animals were galactosemic for up 24 months, almost the entire normal murine life span. As observed in diabetic mice, galactosemia caused the number of endothelial cells and pericytes to decrease over time in the wild-type animals. After 22 months of galactosemia, the number of endothelial cells decreased 25% in wild-type mice vs. age-matched euglycemic wild-type controls (P<0.005); the 22-month galactosemic ICAM-1–/– and CD18–/– mice were largely protected against this loss (Fig. 1
). Compared with age-matched normal controls, endothelial cell counts were reduced by only 19% (P<0.001) in galactosemic ICAM-1–/– mice and were essentially normal in CD18–/– mice. After 22 months, the number of pericytes was decreased by 55% in galactosemic wild-type mice (P<0.0001 vs. age-matched euglycemic wild-type controls). In contrast, no significant decrease in pericytes was observed in the 22-month ICAM-1–/– or CD18–/– galactosemic mice (P>0.05 vs. euglycemic wild-type controls).
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Acellular capillary data in the galactosemic mice closely mirrored those of the diabetic mice. For wild-type C57/BL6 mice, 22 months of galactosemia resulted in an 8-fold increase in acellular capillaries. In contrast, the 22-month galactosemic ICAM-1–/– and CD-18–/– mice developed 56% and 60% fewer acellular capillaries, respectively (P<0.005).
CONCLUSIONS
These data demonstrate that the reduced expression of ICAM-1 or its leukocyte counter-receptor CD18 significantly inhibits the formation of acellular capillaries in experimental models of diabetic retinopathy. Leukocyte adhesion to the diabetic vascular endothelium can promote receptor-mediated endothelial apoptosis via a Fas/FasL mechanism. The reduction in acellular capillaries in diabetic ICAM–/– and CD18–/– mice is a direct measure of the protective effect of inflammation suppression on cell death in the vasculature. Surprisingly, inhibition of leukocyte adhesion also prevented the loss of pericytes, which typically are not in direct contact with adherent leukocytes in the vasculature. We hypothesize that acellular capillaries are the eventual product of chronic, repetitive leukocyte-mediated injury to the vasculature. The damage begins early in diabetes; with time, portions of the vasculature become irreparable as the vascular cells reach replicative senescence.
The long-term wild-type diabetic animals in this study manifested blood-retinal barrier breakdown, one of the most important functional pathologies in diabetic retinopathy. Blood-retinal barrier breakdown was almost totally suppressed in long-term diabetic ICAM-1–/– and CD18–/– animals (Fig. 2
). Many mechanisms may be responsible for this effect and include 1) direct leukocyte-mediated endothelial cell injury and death, 2) leukocyte-mediated injury of the supportive cells (e.g., pericytes); 3) leukocyte-induced opening of tight junctions, and 4) the release of permeability factors such as VEGF from leukocytes, which can directly affect the tight junctions and fenestrae of the endothelial barrier. In conclusion, these data demonstrate that ICAM-1- and CD18-mediated leukocyte adhesion is increased in the retinal vasculature of mice with long-term diabetes or experimental elevation of blood hexose (galactosemia) and accounts for many of the signature lesions of diabetic retinopathy. The data identify ICAM-1 and CD18 as potential therapeutic targets, which in combination with adequate glycemic control may be used to manage the vision-threatening sequelae of diabetes.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1476fje; 
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