In the present study we analyzed the levels of two major endogenous inhibitors of angiogenesis, TSP1 and PEDF, in the vitreous samples from patients with diabetes compared with those from controls. Our hypothesis is that decreased production of TSP1 occurs during diabetes and contributes to the development and progression of diabetic retinopathy. We observed variation in the levels of TSP1 in vitreous samples prepared from diabetic patients compared to control. This ranged from minimal changes in TSP1 levels to little or no detectable TSP1 in vitreous samples from diabetic patients. In contrast, we detected significant levels of PEDF in all vitreous samples with minimal effects on PEDF level with diabetes. However, we observed two major isoforms of PEDF in human vitreous samples, which migrated differently on SDS-PAGE. We observed that TSP1 level was consistently lower in vitreous samples prepared from diabetic patients with higher molecular weight isoform of PEDF. Thus, the neuro-protective and/or antiangiogenic activity of PEDF, rather than its level, may contribute to the development and progression of diabetic retinopathy. The exact identity and the neuro-protective and/or antiangiogenic activity of these isoforms are subject of future investigation.
The tight regulation of angiogenesis is achieved by a balanced production of positive and negative factors. Alterations in this balance, under various pathological conditions such as diabetes, result in angiogenesis. Vascular endothelial growth factor (VEGF) acts as an EC mitogen in vitro and promotes vascular permeability and angiogenesis in vivo (29
). Intraocular VEGF concentrations are increased during the periods of active intraocular neovascularization in patients with PDR (2
). However, little is known about the potential changes in expression and/or activity of endogenous inhibitors of angiogenesis during diabetes.
Many endogenous inhibitors of angiogenesis, including TSP1, and its closely related family member TSP2, angiostatin, endostatin, PEDF, interferon-α/β, and platelet factor 4 have been reported (32
). TSP1 was the first endogenous inhibitor of angiogenesis identified whose expression is suppressed during progression of many solid tumors is associated with the activation of angiogenic switch (7
). We consider TSP1 to be an angiogenic inhibitor associated with the development and progression of diabetic retinopathy (8
). TSP1 is present in vitreous and aqueous humor at high levels and produced by almost all known cell types in the eye including retinal endothelial cells, astrocytes, pericytes, and elsewhere including retinal pigmented epithelial cell, corneal epithelial and endothelial cells, and trabecular meshwork cells (10
). It specifically inhibits EC proliferation and migration, and blocks angiogenesis and tumor growth (34
). TSP1 and its antiangiogenic peptides effectively inhibit new blood vessel growth during OIR (9
). However, the possibility that administration of TSP1 and/or its antiangiogenic peptides may inhibit the development and progression of PDR needs evaluation. Production of TSP1 and TSP2 by astrocytes are recently shown to be essential for appropriate synaptogenesis and retinal neurons functions (41
). Thus, alterations in TSP1 levels may also impact retinal neuronal functions contributing to visual dysfunctions associated with diabetes (42
). Furthermore, decreased production of TSP1, as well as PEDF and endostatin, observed in eyes with age-related macular degeneration (38
) suggest an important role for these angiogenesis inhibitors in modulation of choroidal vascular homeostasis.
In diabetic retinopathy, the total protein concentration of vitreous is generally increased perhaps due to the loss of retinal EC barrier function, an early dysfunction associated with diabetes. Since changes in total vitreous protein content is a likely concern, an important early characteristic of diabetic retinopathy, we utilized volumes of vitreous for our analysis rather that total protein concentration in order to normalize across all samples. In addition, to circumvent the possibility of vitreous hemorrhage affecting our results, we removed cell debris and platelets, a major source of TSP1, before further analysis of the samples. However, this does not rule out the potential release of TSP1 from activated platelets resulting in increased levels of TSP1. Although obtaining vitreous samples from patients in early stages of diabetic retinopathy may be challenging, it will help to address these concerns. Alternatively, one can evaluate TSP1 levels following laser treatment and quiescence of retinal vasculature. However, the limited changes observed in vitreous levels of PEDF suggest a minimal contribution from contaminating serum.
TSP1 concentration was unaffected in some diabetic patients and was dramatically down regulated in others. Since we didn't further classify the patients with diabetes into active (highly active neovascularization with rapidly proliferative membrane and fresh vitreous hemorrhage or retinal detachment) or quiescent (chronic neovascularization with extensive panretinal laser photocoagulation and minimal background retinopathy), there are three possible explanations for the results seen here. First, diabetic retinopathy was active in patients with low TSP1 in the vitreous fluid. Second, diabetic retinopathy may be quiescent in patients with a high level of TSP1 in the vitreous fluid. Third, retinopathy may be active or inactive when TSP1 is high or low, respectively, depending on the concentration of stimulators such as VEGF. Thus, the activity of diabetic retinopathy rather than its severity more accurately reflects the effects of angiogenic stimulators and inhibitors. Unfortunately, the lack of sufficient patient information was prohibitory in delineating these possibilities. Further investigation of VEGF levels and better classification retinopathies are needed for more accurate assessment of the TSP1 changes during diabetes and its impact on the development and progression of diabetic retinopathy.
PEDF is also a potent inhibitor of ocular angiogenesis (13
). Alterations in PEDF levels in patients with diabetes compared with nondiabetic patients have been controversial and not clearly resolved. It was reported that the level of PEDF in vitreous was lower in patient with diabetes (20
). There are also reports that PEDF concentration was higher in the vitreous from patients with diabetes (22
). Our study showed that PEDF was at consistently high levels in the eyes of diabetic and control groups. However, we detected two different isoforms of PEDF in human vitreous samples with different migration/molecular weight (). One isoform migrated faster than other isoform on SDS-PAGE (L, lower molecular weight). We consistently observed lower levels of TSP1 in patient with higher molecular weight PEDF isoform.
Multiple polymorphisms have been reported in human PEDF gene (27
) and an association between some of these polymorphisms (the promoter and exon 3 coding region) and diabetic retinopathy and age-related macular degeneration have been reported (25
). However, it is not known whether these polymorphisms result in production of proteins with different molecular weight or expression level. The identity and the level of transcripts generated by these polymorphisms, and the identity/activity of their potential product require further investigation. A potential polymorphism reported in intron 5 (just upstream from the splice acceptor site) may be a potential candidate for generation of an alternatively spliced transcript producing a different size protein (27
). Thus, the identity of the PEDF polymorphisms and their protein products, and their association with the severity of diabetic retinopathy, will be very informative. Furthermore, this may allow the development of a screening method for identification of diabetic patients that are at greater risk for the development of PDR.
TSP1 and PEDF, as endogenous inhibitors of angiogenesis, exhibit different expression patterns and functions in retinal vascular development and angiogenesis (4
). Although the level of PEDF was not dramatically changed in the eyes of patients with diabetes the involvement of different PEDF isoform in the pathogenesis of diabetic retinopathy needs further investigation, especially in regards to its effect on TSP1 expression. It is possible that different isoforms of PEDF have different antiangiogenic potential during development and progression of diabetic retinopathy (26
), perhaps through modulation of TSP1 level. Although vitamin A is shown to increase expression of TSP1 and PEDF in retinal pigmented epithelial cells (46
) nothing is known about the regulation of TSP1 by PEDF. Thus, further investigations are needed to address the relationship of proangiogenic factors such as VEGF and, isoforms of PEDF and TSP1 expression in the pathogenesis of diabetic retinopathy.
In summary, we demonstrated that the vitreous TSP1 levels varied among diabetic samples and was consistently lower in the diabetic patients which expressed the higher molecular weight isoform of PEDF. The vitreous levels of PEDF, however, did not vary significantly among diabetic and control patients. Thus, decreased production of TSP1, along with expression of the high molecular weight isoform of PEDF, in patients with diabetes may indicate a greater risk for development of severe retinopathies.