Beneficial effects of fenofibric acid on overexpression of extracellular matrix components, COX-2, and impairment of endothelial permeability associated with diabetic retinopathy

doi:10.1016/j.exer.2015.08.010

Experimental Eye Research

Volume 140, November 2015, Pages 124-129

https://doi.org/10.1016/j.exer.2015.08.010 Get rights and content

Highlights

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Mechanistic insight into fenofibric acid's beneficial effect in DR.
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Fenofibric acid prevents high glucose-induced ECM overexpression in endothelial cells.
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Fenofibric acid reduces retinal inflammation associated with DR.

Abstract

In the Fenofibric Acid (FA) Intervention and Event Lowering in Diabetes (FIELD) study, FA, a lipid-lowering drug, has been shown to significantly reduce macular edema in diabetic patients. In the present study, we investigated whether FA reduces vascular permeability by inhibiting cyclooxygenase-2 (COX-2), a critical mediator of inflammation, and reducing overexpression of fibronectin (FN) and collagen IV (Coll IV), two basement membrane (BM) components upregulated in diabetic retinopathy. Rat retinal endothelial cells (RRECs) were grown in normal (N:5 mM glucose) or high glucose (HG:30 mM glucose) medium with or without FA for 7 days. Total protein isolated from these cells was assessed for FN, Coll IV, COX-2, and zonula occludens-1 (ZO-1), a tight junction protein, using Western blot analysis. In addition, the distribution and localization of ZO-1 was determined by immunofluorescence microscopy, and cell monolayer permeability was studied by in vitro permeability (IVP) assay. RRECs grown in HG medium showed significant increase in FN, Coll IV, and COX-2 expression (179%, 144%, 139% of N respectively), and a decrease in ZO-1 expression (48% of N) compared to those of N cells. Cells grown in HG medium supplemented with FA significantly reduced FN, Coll IV, and COX-2 expression by 47%, 32%, and 34% respectively, with concomitant increase in ZO-1 expression by 42%. In parallel studies, IVP assays showed a significant increase (139% of N) in cell monolayer permeability in RRECs grown in HG medium, which was significantly reduced with FA treatment. Additionally, immunostaining results indicated FA prevents HG-induced downregulation of ZO-1. The findings indicate that the beneficial effect of FA in reducing excess permeability is mediated, at least in part, by downregulating abnormal overexpression of BM components and inflammatory factors and preventing compromised tight junctions associated with diabetic retinopathy.

Section snippets

Background

FA is a lipid lowering drug that has been shown to be effective in reducing the risk of developing cardiovascular disease events (Keech et al., 2005). There is now consistent evidence from two major trials, the FA Intervention and Event Lowering in Diabetes (FIELD) study (Keech et al., 2007) and the Action to Control Cardiovascular Risk in Diabetes Eye (ACCORD-Eye) study (Chew et al., 2010) that FA reduces the risk of progression of diabetic retinopathy. In the FIELD study, which involved an

Cell culture – rat retinal endothelial cells

Rat retinal endothelial cells (RRECs) ascertained positive for von Willebrand factor were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS (Hyclon, Thermo Scientific, Waltham, MA), antibiotics, and antimycotics. Second to fourth passage cells were used in this study. All experiments were repeated at least four times. To examine the effect of FA on retinal vascular BM components, FN and Coll IV expression, and ZO-1 and COX-2 expression, RRECs were grown in normal medium (5 mmol/l

Fenofibric acid abrogates fibronectin and collagen IV overexpression induced by HG condition

Western blot analysis showed significantly increased FN protein expression in RRECs grown in HG medium when compared to those grown in normal medium (179 ± 23% of normal, p < 0.001, n = 6). When RRECs grown in HG medium were treated with FA, a significant reduction in FN protein level was observed compared to untreated RRECs grown in HG medium (132 ± 14% of normal vs. 179 ± 23% of normal, p = 0.001, n = 6) (Fig. 1A and B).

In parallel experiments, Western blot analysis showed significantly

Discussion

While substantial clinical benefits with FA on diabetic retinopathy were observed in the FIELD (Keech et al., 2007) and ACCORD-Eye studies (Chew et al., 2010), the underlying therapeutic mechanisms are not yet clear. In this study, we investigated the effect of FA on the inner BRB and how it may contribute to the maintenance of vascular integrity under HG condition. Our findings indicate that FA treatment prevents increased retinal endothelial cell monolayer permeability induced by the HG

Acknowledgments

Research was supported by National Institutes of Health/National Eye Institute Grants EY014702 and EY025528, and in part by a departmental grant from the Massachusetts Lions Organization (SR). In addition, this study was supported by grants from Ministerio de Ciencia y Tecnología (SAF2012-35562) and CIBER for Diabetes and Associated Metabolic Diseases (CIBERDEM). CIBERDEM is an initiative of the Instituto de Salud Carlos III. We acknowledge the assistance of Solvay Pharma S.A. in providing FA.

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2022, Handbook of Basic and Clinical Ocular Pharmacology and Therapeutics
The magnitude of the healthcare challenges of diabetic retinopathy are widely appreciated and drug therapy is used in various ways to reduce the burden of vision loss in patients with diabetes. The different approaches may be organized in a variety of ways, but here a discussion of systemic therapy will be followed by a consideration of local delivery. In both instances, an attempt will be made to map individual therapies to what we know of the key elements of the pathogenesis of diabetic retinopathy, although interestingly, there is significant overlap between modes of actions for several of the classes of drug to be considered.
Retinal capillary basement membrane thickening: Role in the pathogenesis of diabetic retinopathy
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Citation Excerpt :
Studies conducted from our lab have demonstrated that fenofibric acid can reduce HG-induced fibronectin and collagen overexpression (Roy et al., 2015; Trudeau et al., 2011) concomitant with a decrease in cyclooxygenase-2, a pro-inflammatory molecule, as well as improving tight junction functionality by upregulating zonula occludens-1 (ZO-1) expression (Roy et al., 2015). Additionally, retinal endothelial cells grown in HG condition exhibited increased cell monolayer permeability whereas cells grown in HG and treated with fenofibric acid showed reduced cell monolayer permeability, indicating fenofibric acid's beneficial effects against vascular permeability (Roy et al., 2015). Moreover, in an in vivo environment, oral administration of fenofibrate halted retinal capillary BM thickening, downregulated VEGF and ROS expression, and prevented retinal vascular permeability in diabetic rats (Li et al., 2018), suggesting that fenofibrate can preserve BM functionality and prevent retinal vascular leakage associated with DR. Taken together, while further studies are necessary to better characterize fenofibrate efficacy and other gene modulatory strategies in human DR, targeting abnormal vascular BM thickening holds promise amidst the growing need for therapeutic strategies to treat DR.
Vascular basement membrane (BM) thickening has been hailed over half a century as the most prominent histological lesion in diabetic microangiopathy, and represents an early ultrastructural change in diabetic retinopathy (DR). Although vascular complications of DR have been clinically well established, specific cellular and molecular mechanisms underlying dysfunction of small vessels are not well understood. In DR, small vessels develop insidiously as BM thickening occurs. Studies examining high resolution imaging data have established BM thickening as one of the foremost structural abnormalities of retinal capillaries. This fundamental structural change develops, at least in part, from excess accumulation of BM components. Although BM thickening is closely associated with the development of DR, its contributory role in the pathogenesis of DR is coming to light recently. DR develops over several years before clinical manifestations appear, and it is during this clinically silent period that hyperglycemia induces excess synthesis of BM components, contributes to vascular BM thickening, and promotes structural and functional lesions including cell death and vascular leakage in the diabetic retina. Studies using animal models show promising results in preventing BM thickening with subsequent beneficial effects. Several gene regulatory approaches are being developed to prevent excess synthesis of vascular BM components in an effort to reduce BM thickening. This review highlights current understanding of capillary BM thickening development, role of BM thickening in retinal vascular lesions, and strategies for preventing vascular BM thickening as a potential therapeutic strategy in alleviating characteristic lesions associated with DR.
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Recent large-scale studies have shown that fenofibrate is able to stop the progression of DR [83–85]. Fenofibrate has been shown to downregulate the abnormal overexpression of basement membrane components and inflammatory mediators such as NF-κB and VEGF [86,87]. Fenofibrate can also reduce hyperglycaemia-induced oxidative stress in rat retinas and reduce the expression of thioredoxin-interacting protein (TXNIP) which is known to reduce cellular antioxidant capacity [88].
Diabetic retinopathy (DR) is a microvascular complication of diabetes and is the leading cause of vision loss in people with diabetes. The current treatments do not target early stages of disease or impede disease progression. Therefore, the identification of new therapeutic targets, the development of novel therapies targeting early stages of the disease and accurate models that simulate pathological characteristics of this disorder are crucial. This review provides an overview of the pathological mechanisms underlying the development of DR, highlighting the recent advances in current and emerging treatments for DR.
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One might speculate that since the minority of patients had a complete absence of fluorescein leakage even after anti-VEGF therapy, some aspect of vessel permeability may be mediated by VEGF-independent pathways. A few studies have suggested that other signaling pathways such as angiopoietin-2,24 cyclooxygenase,25,26 and protein kinase Cζ27 may play a role in vessel leakage in DR. In the RIDE and RISE studies, 36 months of ranibizumab treatment was associated with long-term improvement of DR severity.13,14
To investigate the clinical importance of changes in diabetic retinopathy severity score (DRSS) in patients with diabetic macular edema (DME) treated with intravitreal ranibizumab.
Post hoc analysis of the phase III RIDE and RISE studies of ranibizumab for treatment of DME.
Four hundred sixty-eight eyes treated with ranibizumab from randomization with gradable DRSS on baseline fundus photographs.
Visual and anatomic outcomes were examined in eyes grouped according to DRSS change from baseline to month 24.
Mean best-corrected visual acuity (BCVA) letter score change, proportion of patients with 15 or more Early Treatment Diabetic Retinopathy Study (ETDRS) letter score change, mean contrast sensitivity change, proportion of patients with resolved macular edema, and leakage on fluorescein angiography.
Most (56.8%) patients treated with ranibizumab experienced 1-step or more improvement in DRSS from baseline to month 24; 40.0% had no change, and 3.2% experienced DRSS worsening. Patients with DRSS stability or improvement had greater mean BCVA letter score changes (+15.1, +14.2, +11.3, and +11.2 letters for ≥3-step improvement, ≥2-step improvement, 1-step improvement, and no DRSS change, respectively) compared with +5.0 letters in patients who had any DRSS worsening. Best-corrected visual acuity letter score gain of 15 letters or more was more common in patients with 2-step or 3-step or more DRSS improvement (51.9% and 44.6%, respectively) compared with those with a 1-step DRSS improvement, no change, or worsening (37.9%, 39.6%, and 26.7%, respectively). A loss of 15 letters or more in BCVA was more common in patients with any DRSS worsening (13.3%) compared with patients who had stable or improved DRSS (0%–2.8%). Resolution of macular edema was more common in patients with DRSS improvement: 84.2%, 87.7%, and 92.3% of patients with 1-step, 2-step or more, and 3-step or more improvement in DRSS achieved central foveal thickness of 250 μm or less, compared with 65.2% and 53.3% of patients who had no DRSS change or any DRSS worsening.
These findings provide further support that improvement in DRSS is a clinically important outcome that should be evaluated as a measure of treatment effectiveness in future studies of diabetic eye disease.
Mechanistic Insights into Pathological Changes in the Diabetic Retina: Implications for Targeting Diabetic Retinopathy
2017, American Journal of Pathology
Citation Excerpt :
At high doses, cyclooxygenase-2 inhibitor meloxicam has been shown to reduce endothelial NO synthase concentrations,62 NF-κB activation levels,37 and leukocyte adhesion62 in diabetic retinas. In addition, our study indicates that fenofibrate can reduce high glucose-induced cyclooxygenase-2 up-regulation to exhibit anti-inflammatory effects.65 Aspirin has also been shown to significantly reduce the adhesiveness of leukocytes62 and, at high doses, to minimize the development of microvascular lesions in patients in non-proliferative DR.64,66,67
Increasing evidence points to inflammation as one of the key players in diabetes-mediating adverse effects to the neuronal and vascular components of the retina. Sustained inflammation induces biochemical and molecular changes, ultimately contributing to retinal complications and vision loss in diabetic retinopathy. In this review, we describe changes involving metabolic abnormalities secondary to hyperglycemia, oxidative stress, and activation of transcription factors, together with neuroglial alterations in the diabetic retina. Changes in biochemical pathways and how they promote pathophysiologic developments involving proinflammatory cytokines, chemokines, and adhesion molecules are discussed. Inflammation-mediated leukostasis, retinal ischemia, and neovascularization and their contribution to pathological and clinical stages leading to vision loss in diabetic retinopathy (DR) are highlighted. In addition, potential treatment strategies involving fibrates, connexins, neuroprotectants, photobiomodulation, and anti-inflammatory agents against the development and progression of DR lesions are reviewed. The importance of appropriate animal models for testing novel strategies against DR lesions is discussed; in particular, a novel nonhuman primate model of DR and the suitability of rodent models are weighed. The purpose of this review is to highlight our current understanding of the pathogenesis of DR and to summarize recent advances using novel approaches or targets to investigate and inhibit the retinopathy.
Dual Anti-Inflammatory and Anti-Angiogenic Action of miR-15a in Diabetic Retinopathy
2016, EBioMedicine
Activation of pro-inflammatory and pro-angiogenic pathways in the retina and the bone marrow contributes to pathogenesis of diabetic retinopathy. We identified miR-15a as key regulator of both pro-inflammatory and pro-angiogenic pathways through direct binding and inhibition of the central enzyme in the sphingolipid metabolism, ASM, and the pro-angiogenic growth factor, VEGF-A. miR-15a was downregulated in diabetic retina and bone marrow cells. Over-expression of miR-15a downregulated, and inhibition of miR-15a upregulated ASM and VEGF-A expression in retinal cells. In addition to retinal effects, migration and retinal vascular repair function was impaired in miR-15a inhibitor-treated circulating angiogenic cells (CAC). Diabetic mice overexpressing miR-15a under Tie-2 promoter had normalized retinal permeability compared to wild type littermates. Importantly, miR-15a overexpression led to modulation toward nondiabetic levels, rather than complete inhibition of ASM and VEGF-A providing therapeutic effect without detrimental consequences of ASM and VEGF-A deficiencies.

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Beneficial effects of fenofibric acid on overexpression of extracellular matrix components, COX-2, and impairment of endothelial permeability associated with diabetic retinopathy

Highlights

Abstract

Section snippets

Background

Cell culture – rat retinal endothelial cells

Fenofibric acid abrogates fibronectin and collagen IV overexpression induced by HG condition

Discussion

Acknowledgments

Atherosclerosis

Eur. J. Pharmacol.

Vasc. Pharmacol.

Lancet

Biochem. Biophys. Res. Commun.

Am. J. Pathol.

Lancet

Exp. Eye Res.

Lancet

Prog. Retin. Eye Res.

A study of capillary pericyte viability on extracellular matrix produced by endothelial cells in high glucose

Diabetologia

Pericyte adhesion is impaired on extracellular matrix produced by endothelial cells in high hexose concentrations

Diabetologia

Significance of COX-2 expression in human renal cell carcinoma cell lines

Int. J. Cancer

Therapeutic effects of PPARalpha agonists on diabetic retinopathy in type 1 diabetes models

Diabetes

Tight glycemic control regulates fibronectin expression and basement membrane thickening in retinal and glomerular capillaries of diabetic rats

Investig. Ophthalmol. Vis. Sci.

Effects of medical therapies on retinopathy progression in type 2 diabetes

N. Engl. J. Med.

Endothelin receptor blockade prevents augmented extracellular matrix component mRNA expression and capillary basement membrane thickening in the retina of diabetic and galactose-fed rats

Diabetes

Fenofibrate prevents the disruption of the outer blood retinal barrier through downregulation of NF-κB activity

Acta Diabetol.

Advanced glycation end products: sparking the development of diabetic vascular injury

Circulation