Findings from the present study indicate that FA treatment prevents increased RPE permeability induced by HG + IL-1β and that this beneficial effect of FA is associated with decreases in HG- and HG + IL-1β-induced FN and Coll IV overexpression. This suggests that FA can prevent the breakdown of BRB permeability at least in part by normalizing ECM protein overproduction. In addition, we confirmed previous reports showing that the altered amount of tight junction proteins was not necessarily the only factor regulating tight junction functionality and that the distribution of the tight junction proteins plays an important role in barrier permeability.23,24
In fact, the protective effect of FA on RPE disruption induced by HG + IL-1β is in part mediated by its ability to prevent the aberrant distribution of tight junction proteins. The capacity of FA in maintaining the tight junction distribution and its suppressive effect on ECM overproduction could be involved in the beneficial effects of fenofibrate on DME. However, further investigation to determine the mechanisms by which FA affects ECM protein expression and tight junction protein distribution are needed.
Importantly, our findings from this study implicate a downregulation effect of FA on extracellular matrix protein levels, which could play a role in preventing vascular permeability and in underscoring the importance of FN and Coll IV in forming a selective permeable outer BRB. In this regard we have previously shown that reducing basement membrane thickening by downregulating extracellular matrix components including FN and Coll IV is effective in preventing the apoptosis and increased permeability associated with DR.11,25
Additionally, studies on RPE monolayers cultured on laminin-coated filters indicated that extracellular matrix components promote RPE morphology and the formation of a selective permeability barrier to various tracers.26
Increased levels of proinflammatory cytokines play a key role in the pathogenesis of DME.17,27,28
Treatment of RPE cells with either serum, interferon-γ, tumor necrosis factor-α, hepatocyte growth factor (HGF), interleukin (IL)-1β or placental growth factor-1 increases permeability and alters the expression or content of tight junction molecules.23,29–31
Because IL-1β plays an important role in the development of DR,32–34
we decided to use the cytokine together with HG conditions to mimic the diabetic milieu. A significant overexpression of FN and Coll IV was observed after treating ARPE-19 cells with IL-1β in the presence of HG, and this overexpression was associated with an increase in permeability. Overall, these findings indicate that a higher content of basement membrane components may contribute to the impairment of barrier function, leading to excess permeability. In addition, the overexpression of basement membrane components known to be induced by inflammatory cytokines such as IL-1β35,36
may be involved in hyperpermeability, which occurs in DR.
Microvascular basement membrane is an important component of the blood barrier system, which participates in the regulation of vascular permeability. Thus, any changes to the basement membrane structure or its composition may adversely affect its function. Previous studies demonstrated the ability of fenofibrate to decrease extracellular matrix accumulation in renal cortex of streptozotocin-induced diabetic rats37
and in kidneys of spontaneously hypertensive rats.38
In addition, fenofibrate treatment was shown to affect extracellular matrix changes associated with systolic failure seen in ascending aortic constriction in chronic pressure overload mice.39
Our results from this study parallel these findings and demonstrate fenofibrate treatment's beneficial effects on pathologic changes associated with the overexpression of extracellular matrix proteins.
The exact cellular mechanisms by which FA influences extracellular matrix component levels is unclear. Recent studies have focused on the ability of FA to activate peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor that regulates the genes involved in cellular lipid catabolism. The activation of PPARα increases lipolysis and the elimination of triglyceride-rich particles from plasma and also increases the synthesis of apoproteins, which leads to a reduction in very low-density and low-density fractions and an increase in the high-density lipoprotein fraction containing apoprotein. PPARα may regulate extracellular matrix turnover through consequently inhibiting matrix metalloproteinases38,39
or decreasing plasminogen activator inhibitor-1.37
However, the exact pathway involving PPARα and its downstream effectors has not been completely defined.
Other studies have investigated how fenofibrate may suppress oxidative stress and MAPK activation, thus decreasing TGF-β levels and ultimately affecting extracellular matrix accumulation.38
Finally, one cannot rule out other mechanisms whereby fenofibrate may affect vascular permeability. One report demonstrated that fenofibrate is able to reduce apoptosis in human retinal endothelial cells, which is associated with DR.40
The mechanism by which fenofibrate exerted its antiapoptotic effect was found to be AMP-activated protein kinase (AMPK)–dependent and PPARα-independent. Preventing unwanted apoptosis in the retinal vasculature may help maintain vessel integrity and prevent leakage associated with DR. In addition, we have recently shown that RPE disruption induced by IL-1β is prevented by FA because of its ability to suppress AMPK activation.24
This finding indicates that suppression rather than activation of AMPK is the mechanism by which FA prevents the hyperpermeability induced by HG + IL-1β. In the same paper, we reported that AMPK activation in human RPE from diabetic donors was significantly higher than from nondiabetic donors and very similar to that obtained in ARPE-19 cells cultured under high (25 mM) glucose + IL-1β. Taken together, our results suggest that the suppression of AMPK activation is a mechanism by which fenofibrate may prevent or arrest diabetic macular edema.
A limitation of the present study is that it focuses on the effects of FA only on the outer BRB. As such, further studies are needed to investigate the effect of FA on the inner BRB and the contribution of FA on overall BRB breakdown. However, findings from this study documented an important proof of concept that HG-induced excess accumulation of basement membrane components of the outer BRB is involved in increased retinal permeability and that the protective effect of FA against leakage of the outer BRB is at least in part linked to the inhibitory effect of FA on specific basement membrane component expression in the RPE cells. The ability of FA to prevent basement membrane component overexpression may have significance for other diabetic microangiopathies beyond DME.