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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 15, 2002 as doi:10.1096/fj.02-0157fje. |
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2,3,3
2,
* Retina Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA;
Department of Vitreoretinal Surgery, Center for Ophthalmology and Center for Molecular Medicine (ZMMK), University of Köln, D-50931 Köln, Germany;
Beetham Eye Institute, Joslin Diabetes Center, Boston, Massachusetts, USA;
Surgical Research Laboratories, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA; and
|| Dana Faber Cancer Institute and
Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA
2Correspondence: Massachusetts Eye and Ear Infirmary, Harvard Medical School, 243 Charles Street, Boston, MA 02114, USA. E-mail: tony adamis{at}meei.harvard.edu; Department of Vitreoretinal Surgery, Center for Ophthalmology and Center for Molecular Medicine (ZMMK), University of Köln, Joseph Stelzmann Str. 9, D-50931 Köln, Germany. E-mail: JoussenA{at}aol.com
SPECIFIC AIMS
Diabetic retinopathy, a leading cause of blindness, is characterized by early retinal microvascular dysfunction. Increased leukocyte adhesion is causally linked to blood–retinal barrier breakdown and endothelial cell injury and death. However, the molecular mechanisms underlying these two important phenomena and their relationship to one another are not fully understood. In this study, we evaluated the mechanistic role of the Fas/FasL pathway in the pathogenesis of diabetic leukocyte-mediated retinal vascular dysfunction. Our data indicate that the Fas/FasL pathway is operative in the death of the retinal microvasculature and is directly causal for breakdown of the blood–retinal barrier. These data indicate that targeting the Fas/FasL pathway may prove beneficial in treating diabetic retinopathy.
PRINCIPAL FINDINGS
1. Diabetic neutrophils induce endothelial cell apoptosis in vitro through Fas/FasL
To determine whether diabetic leukocytes preferentially induce endothelial cell apoptosis, neutrophils derived from nondiabetic and diabetic animals were evaluated using a leukocyte/endothelial coculture assay. The number of TUNEL-positive endothelial cells per adherent neutrophil increased from 0.23 ± 0.11 for leukocytic populations isolated from nondiabetic rats to 1.48 ± 0.41 (P<0.001), 4.53 ± 1.94 (P<0.001), and 4.46 ± 2.11 (P<0.05) from rats with 1 wk, 6 wk, or 6 months of diabetes, respectively (P<0.001).
To confirm whether Fas and FasL were involved in mediating this increase in apoptosis, endothelial cells were preincubated for 1 h with neutralizing antibodies against Fas, DR4, and DR5. Conversely, neutrophils were preincubated with anti-FasL or anti-TRAIL antibodies. Endothelial cell pretreatment with neutralizing anti-Fas antibodies reduced TUNEL staining in 6 month diabetic animals by 68% from an average 4.25 ± 1.92 to 1.42 ± 0.71 apoptotic endothelial cells per adherent leukocyte (P<0.0005). Pretreatment of the neutrophils with anti-FasL antibody resulted in an 81.3% reduction to 0.84 ± 0.38 (P<0.0001). In contrast, pretreatment of endothelial cells with anti-DR4/DR5 or pretreatment of neutrophils with anti-TRAIL did not significantly reduce apoptosis.
2. Diabetes increases Fas and FasL expression in vivo
To investigate whether diabetes-induced expression of FasL on leukocytes provided a source of initiation of Fas/FasL-mediated apoptosis, leukocyte cell surface expression of FasL and TRAIL was determined by FACS analysis. Neutrophils were isolated from nondiabetic rats and rats with diabetes of 1 wk or 6 month duration. Expression of FasL and TRAIL was not significantly different from control antibody in nondiabetic animals (4.2±2.7%). In contrast, neutrophil FasL expression increased to 28.5 ± 3.6% (P<0.005) after 1 wk of diabetes and to 36.2 ± 4.3% (P<0.001) after 6 months of diabetes. The expression of TRAIL did not significantly change even after 6 months of disease.
To determine whether diabetes increased Fas expression in the retina and if this expression colocalized with retinal vascular endothelial cells, Fas expression was evaluated using immunohistochemistry in paraffin sections derived from 2 wk diabetic and control rats. Retinal endothelial cells were identified using an antibody to CD31. Fas staining was substantially increased in diabetic rats compared with nondiabetic rats, where staining was minimal. CD31 staining of endothelial cells was evident in both diabetic and nondiabetic animals. The majority of diabetes-induced Fas staining colocalized with areas of CD31 staining.
3. Leukocytes mediate Fas-induced retinal endothelial cell injury, apoptosis, and death in vivo
To investigate the in vivo role of Fas and FasL in mediating leukocyte-induced retinal endothelial cell death, the effect of a neutralizing anti-FasL antibody on retinal endothelial injury was assessed using propidium iodide (PI) incorporation (Fig. 1
). As previously demonstrated, diabetic retina exhibited a marked increase in PI-labeled cells, located mainly in clusters adjacent to adherent leukocytes (P<0.0005). In contrast, the number of PI-labeled endothelial cells in the eyes of animals treated with neutralizing anti-FasL antibody was suppressed in the retinal arterioles (n=14, 2.78±2.00 vs. 32.0±8.5; P<0.0001), venules (n=14, 3.28±3.14 vs. 36.35±9.01; P<0.0001), and capillaries (n=14, 4.92±4.69 vs. 27.78±8.65; P<0.0001) compared with animals receiving control antibody.
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To identify if and localize where apoptosis was occurring under these conditions, formalin-fixed paraffin-embedded retinal sections were stained using TUNEL and M30 CytoDeath antibody. TUNEL-positive cells showed nuclear staining whereas M30-positive cells stained the cytoplasm. In diabetic animals, TUNEL and M30 staining was readily observed and positive cells were located predominantly in the ganglion cell layer and the vascular endothelium. When retinal vessels were evident in these routine histological sections, there was associated staining. In contrast, nondiabetic animals showed little staining anywhere in the retina. When diabetic animals were treated with anti-FasL antibody, TUNEL and M30 staining was attenuated.
To quantitate apoptotic cell death in the retina, a modified ELISA for fragmented DNA was used. After 2 wk of diabetes, fragmented DNA had increased by almost 13.84 ± 0.41-fold (P<0.005) compared with nondiabetic retina (1.0±0.15-fold). Systemic treatment with anti-FasL antibody reduced fragmented retinal DNA to nearly nondiabetic levels (1.22±0.82-fold; P<0.0005) whereas control IgG antibody had no significant attenuating effect (14.64±0.64-fold).
4. Fas-FasL blockade does not decrease leukocyte adhesion in vivo
To determine whether the effects observed using neutralizing FasL antibody were due to inhibition of leukocyte adhesion in the retinal vasculature of diabetic animals, we used concanavalin A lectin staining in retinal flat mounts. Leukocyte adhesion in vivo was not significantly altered by control or anti-FasL antibody compared with diabetic control animals when assessing arterioles (n=13; 69.84±7.35 vs. 51.3±8.24; P>0.05), venules (n=14; 60.15±8.96 vs. 45.64±12.64; P>0.05), or capillaries (n=14; 60.23±14.26 vs. 47.63±9.25; P>0.05). A slight reduction of adherent leukocytes by anti-FasL was observed in arterioles only when compared with IgG control. Adherence was reduced to values observed in the untreated diabetic animals and was still far above nondiabetic values.
5. Role of Fas-Fas ligand in diabetes-induced blood–retinal barrier breakdown in vivo
To determine whether the Fas/FasL system was involved in mediating diabetes-induced ocular microvascular complications, retinal vascular permeability in nondiabetic and 2 wk diabetic rats was assessed 48 h after treatment with control or anti-FasL neutralizing antibody (Fig. 2
). Two weeks of diabetes increased retinal vascular permeability 2.6-fold (P<0.0001). Systemic treatment with anti-FasL neutralizing antibody reduced retinal vascular permeability in the diabetic retina by 56.5% (n=14; 1.33±0.19 arbitrary units; P<0.0005) vs. diabetic animals alone (n=10; 2.88±0.22) or diabetic animals treated with control antibody (n=14; 3.06±0.06; P<0.0001).
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CONCLUSIONS
Increased leukocyte adhesion has been shown to correlate spatially and temporally with endothelial cell death in the diabetic rat retina. The probable involvement of leukocytes in initiating Fas-mediated retinal endothelial cell death under diabetic conditions is supported by several observations. Our data indicate that the surface expression of FasL on circulating leukocytes is increased in the diabetic state and that these leukocytes are capable of inducing Fas-mediated endothelial cell injury, apoptosis, and blood–retinal barrier breakdown. Incubation of diabetic leukocytes with retinal endothelial cell cultures resulted in marked increase in TUNEL staining that was blocked by either leukocyte pretreatment with anti-FasL or endothelial cell pretreatment with anti-Fas. Increased leukocyte adhesion was associated with endothelial cell injury and apoptosis in vivo that was suppressed by anti-FasL therapy. Neutralization of FasL in vivo did not significantly alter leukocyte adhesiveness to the retinal vascular endothelium, suggesting that although Fas-FasL interactions are responsible for inducing endothelial apoptosis and death, they are not primarily responsible for leukocyte adhesion to the retinal vasculature. According to our data, nonendothelial cells such as retinal ganglion cells and astrocytes were propidium iodide-, M30-, and TUNEL-positive, showing that injury and cell death occur early in the course of diabetes in many different populations. The physiological significance of our findings is substantiated by the inhibition of diabetes-induced blood–retinal barrier breakdown in vivo by neutralizing anti-FasL antibody. Increased blood–retinal barrier breakdown is a major cause of visual loss in diabetes, resulting in diabetic macular edema. Our data suggest that the blood–retinal barrier breakdown that characterizes diabetes may be in part the result of Fas-mediated apoptosis in the vascular endothelium (Fig. 3
).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0157fje; to cite this article, use FASEB J. (November 1, 2002) 10.1096/fj.02-0157fje 
3 Both authors contributed equally to this work.
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