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. 2021 Oct;28(7):e12717.
doi: 10.1111/micc.12717. Epub 2021 May 29.

The role of tumor necrosis factor-α and interferon-γ in the hyperglycemia-induced ubiquitination and loss of platelet endothelial cell adhesion molecule-1 in rat retinal endothelial cells

Randa S Eshaq  1 , Norman R Harris  1
Affiliations

The role of tumor necrosis factor-α and interferon-γ in the hyperglycemia-induced ubiquitination and loss of platelet endothelial cell adhesion molecule-1 in rat retinal endothelial cells

Randa S Eshaq et al. Microcirculation. 2021 Oct.

Abstract

Objective: This study aimed to investigate the role of the hyperglycemia-induced increase in tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in the ubiquitination and degradation of platelet endothelial cell adhesion molecule-1 (PECAM-1) in the diabetic retina.

Methods: Type I diabetes was induced in rats by the injection of streptozotocin, with age-matched non-diabetic rats as controls. Primary rat retinal microvascular endothelial cells were grown in normal or high glucose media for 6 days or in normal glucose media for 24 h with addition of TNF-α and/or IFN-γ. PECAM-1, TNF-α, IFN-γ, and ubiquitin levels were assessed using Western blotting, immunofluorescence, and immunoprecipitation assays. Additionally, proteasome activity was assessed both in vivo and in vitro.

Results: Under hyperglycemic conditions, total ubiquitination levels in the retina and RRMECs, and PECAM-1 ubiquitination levels in RRMECs, were significantly increased. Additionally, TNF-α and IFN-γ levels were significantly increased under hyperglycemic conditions. PECAM-1 levels in RRMECs treated with TNF-α and/or IFN-γ were significantly decreased. Moreover, there was a significant decrease in proteasome activity in the diabetic retina, hyperglycemic RRMECs, and RRMECs treated with TNF-α or IFN-γ.

Conclusion: Tumor necrosis factor-α and IFN-γ may contribute to the hyperglycemia-induced loss of PECAM-1 in retinal endothelial cells, possibly by upregulating PECAM-1 ubiquitination.

Keywords: IFN-γ; PECAM-1; TNF-α; diabetic retinopathy; ubiquitination.

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Figures

Figure 1.
Figure 1.. TNF-α levels under hyperglycemic conditions.
TNF-α levels in retinas, plasma, and RRMECs were determined using western blotting techniques. A) TNF-α levels in retinas collected from diabetic rats were not altered when compared to retinas collected from non-diabetic rats. However, there was a significant increase in TNF-α levels in plasma (B) collected from diabetic rats when compared to controls. C) TNF-α in RRMECs grown under hyperglycemic (HG) conditions had significantly higher levels when compared to RRMECs grown under NG conditions, and no significant change observed with mannitol (Man). Western blotting data is a comparison of the relative TNF-α/β-actin or TNF-α/total protein (Plasma) band densities.*p<0.05, N=5-7 per group in vivo and N=3 per group in vitro.
Figure 2.
Figure 2.. IFN-γ levels under hyperglycemic conditions.
IFN-γ levels were significantly increased in retinas and plasma collected from diabetic rats (A, B) and in RRMECs grown under hyperglycemic conditions (HG) and mannitol (Man) (C) when compared to controls. Western blotting data is a comparison of the relative IFN-γ/β-actin band densities. *p<0.05, N=5-7 per group in vivo and N=3-6 per group in vitro.
Figure 3.
Figure 3.. MMP-2, but not MMP-9, increased TNF-α in RRMECs.
A) TNF-α levels were significantly increased in RRMECs treated with MMP-2, but no change in TNF-α levels (B) was observed in RRMECs treated with MMP-9. C) In silico analysis (Prosper) to predict TNF-α cleavage sites by MMPs revealed cleavage sites for MMP-2, MMP-3, and MMP-9. Western blotting data is a comparison of the relative TNF-α/β-actin band densities.*p<0.05, N=3 per group.
Figure 4.
Figure 4.. TNF-α and IFN-γ significantly decreased PECAM-1 levels in RRMECs.
A) TNF-α (5, 10, 20, 50 ng/ml) treated RRMECs (24 hours) had a significant PECAM-1 loss at 20 and 50 ng/ml. B) TNF-α (20 ng/ml) and IFN-γ (30 ng/ml) treatments for 24 hours significantly decreased PECAM-1 levels in RRMECs, as did hyperglycemia (HG) following 6 days of treatment. C) Representative immunostaining images showing decreased PECAM-1 levels with TNF-α and IFN-γ treatments. D) Quantification of PECAM-1 fluorescence intensity with TNF-α and IFN-γ treatments. Western blotting data is a comparison of the relative PECAM-1/β-actin band densities. Scale bar = 50 μm, *p<0.05, N=3 per group.
Figure 5.
Figure 5.. TNF-α and IFN-γ treatment on RRMECs did not induce apoptosis
A) Apoptosis assays (propidium iodide (PI) and annexin V) of RRMECs treated with TNF-α and IFN-γ showing no change from non-treated (NT) cells. B) Positive controls for apoptosis. C) Quantification of propidium iodide (PI) fluorescence intensity with TNF-α and IFN-γ treatments. D) Quantification of annexin V fluorescence intensity with TNF-α and IFN-γ treatments. Scale bar = 50 μm, *p<0.05, N=3 per group.
Figure 6.
Figure 6.. Total ubiquitination and PECAM-1 ubiquitination levels were significantly increased under hyperglycemic conditions.
Western blot analyses showed a significant increase in total ubiquitination levels in the retina (A) and RRMECs (B) under hyperglycemic conditions and mannitol (Man) when compared to cells grown under normoglycemic (NG) conditions. C) PECAM-1 ubiquitination levels were significantly increased in RRMECs grown under hyperglycemic (HG). Western blotting data is a comparison of the relative ubiquitin/β-actin band densities. *p<0.05, N=11-12 per group in vivo and N=3-6 per group in vitro.
Figure 7.
Figure 7.. TNF-α and IFN-γ treatments significantly increased total and PECAM-1 ubiquitination levels in RRMECs.
A) Total ubiquitination levels in RRMECs treated with TNF-α or IFN-γ were significantly increased. B) PECAM-1 ubiquitination levels were significantly increased with TNF-α or IFN-γ treatments in RRMECs. Western blotting data is a comparison of the relative ubiquitin/β-actin band densities. *p<0.05, N=3 per group.
Figure 8.
Figure 8.. Proteasome activity is decreased under hyperglycemic and inflammatory conditions.
A) Proteasome activity in retinas collected from non-diabetic and diabetic rats expressed as relative fluorescent units (RFU). B) Proteasome activity in RRMECs grown under hyperglycemic conditions, or treated with TNF-α, IFN-γ or both. *p<0.05, N=4-5 per group in vivo and N=3 per group in vitro.
Figure 9.
Figure 9.
A graphical representation of the possible mechanisms involved in hyperglycemia-induced inflammation and PECAM-1 ubiquitination and degradation.
See this image and copyright information in PMC

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