Abstract
Purpose: Endogenous tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) has powerful regulatory effects on inflammation and angiogenesis. In this study, we investigated the role of TIMP-3 in regulating inflammation in the diabetic retina.Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic patients were subjected to Western blot analysis. Streptozotocin-treated rats were used as a preclinical diabetic retinopathy (DR) model. Blood-retinal barrier (BRB) breakdown was assessed with fluorescein isothiocyanate (FITC)-conjugated dextran. Rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by Western blot analysis and ELISA. Adherence of human monocytes to HRMECs was assessed and in vitro angiogenesis assays were performed.Results: Tissue inhibitor of matrix metalloproteinase-3 in vitreous samples was largely glycosylated. Intravitreal injection of TIMP-3 attenuated diabetes-induced BRB breakdown. This effect was associated with downregulation of diabetes-induced upregulation of the p65 subunit of NF-κB, intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor (VEGF), whereas phospho-ERK1/2 levels were not altered. In Müller cell cultures, TIMP-3 significantly attenuated VEGF upregulation induced by high-glucose (HG), the hypoxia mimetic agent cobalt chloride (CoCl2) and TNF-α and attenuated MCP-1 upregulation induced by CoCl2 and TNF-α, but not by HG. TIMP-3 attenuated HG-induced upregulation of phospho-ERK1/2, caspase-3 and the mature form of ADAM17, but not the levels of the p65 subunit of NF-κB and the proform of ADAM17 in Müller cells. TIMP-3 significantly downregulated TNF-α-induced upregulation of ICAM-1 and VCAM-1 in HRMECs. Accordingly, TIMP-3 significantly decreased spontaneous and TNF-α- and VEGF-induced adherence of monocytes to HRMECs. Finally, TIMP-3 significantly attenuated VEGF-induced migration, chemotaxis and proliferation of HRMECs.Conclusion: In vitro and in vivo data point to anti-inflammatory and anti-angiogenic effects of TIMP-3 and support further studies for its applications in the treatment of DR.
Highlights
Diabetic retinopathy (DR) is the most frequent microvascular complication of diabetes mellitus and remains the principal cause of visual impairment among the working-age population
We confirmed that diabetes induced a clear upregulation of the retinal expression of vascular endothelial growth factor (VEGF), a key inducer of diabetes-induced breakdown of the blood-retinal barrier (BRB) (Peach et al, 2018), and we demonstrated that intravitreal Tissue inhibitors of metalloproteinases (TIMPs)-3 administration normalized retinal VEGF expression
We demonstrated that TIMP-3 attenuates upregulation of VEGF in human retinal Müller glial cells induced by the hypoxia mimetic agent CoCl2, HG or the proinflammatory cytokine tumor necrosis factor-α (TNF-α)
Summary
Diabetic retinopathy (DR) is the most frequent microvascular complication of diabetes mellitus and remains the principal cause of visual impairment among the working-age population. In the ocular microenvironment of patients with PDR, several inflammatory and angiogenic factors are upregulated reinforcing the paradigm that inflammation and angiogenesis are critical mechanisms initiating and supporting progression of PDR (Abu El-Asrar et al, 2019, 2021a,b; Rezzola et al, 2020; Wu et al, 2021). Among these factors, vascular endothelial growth factor (VEGF), released in response to hypoxia, is a key player in promoting retinal angiogenesis and vascular leakage (Peach et al, 2018). Effective inhibition of diabetes-induced retinal injury might require multiple agents acting on different pathways to attain complete disruption of disease progression
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