This study evaluated the function of peroxisome proliferator-activated receptor α (PPARα) in vascular endothelial cells (ECs) under physiological and disease conditions. Vascular density and avascular area were evaluated in Griffonia simplicifolia isolectin B4-stained retinas. Endothelial progenitor cells were quantified using flow cytometry. Vascular leakage was evaluated by Evans Blue. The mitochondrial function and morphology were evaluated by a Seahorse analyzer and immunofluorescence staining. Cell senescence was assessed by a senescence-associated β-galactosidase activity assay and Western blot analysis. A significant reduction in the retinal vessel length and vascular mesh density was found in EC-specific PPARα conditional knockout (PPARαECKO) mice. Relative to PPARaflox-KO mice with oxygen-induced retinopathy (OIR), PPARαECKO OIR retina showed enlarged avascular areas and decreased endothelial progenitor cell number, whereas EC-specific PPARα conditional transgenic mice showed reduced avascular areas in the OIR retina. Compared with diabetic PPARaflox-KO mice, diabetic PPARαECKO mice showed declined electroretinographic amplitudes, decreased retinal thickness, and increased retinal vascular leakage. PPARα deficiency exacerbated, whereas PPARα activation alleviated, EC mitochondrial dysfunction induced by diabetic stressors. PPARα-/- ECs developed senescence, prominent oxidant-induced mitochondria fragmentation, and down-regulation of translocase of outer mitochondrial membrane 20 and peroxisome proliferator-activated receptor γ coactivator 1α, relative to wild-type ECs. These results suggest that PPARα in microvascular ECs regulates retinal vascular development and protects ECs against diabetes/hypoxia-induced vascular dysfunction through mitochondrial protective and anti-senescence activities.

Age-related macular degeneration (AMD) and Diabetic retinopathy (DR) are the most common forms of retinal disorders and are the leading causes of vision loss. Traditional treatments such as anti-VEGF therapies often require repeated intravitreal injections. Up to 40 % DR/AMD patients do not adequately respond to the anti-VEGF therapies. Therefore, non-VEGF therapies with alternative mechanisms are needed to address these unmet clinical needs. Fenofibrate (an FDA-approved low-cost oral drug) is a peroxisome proliferator-activated receptor-α (PPARα) agonist. We developed large-sized fenofibrate-loaded biodegradable microparticles capable of achieving high drug loading (up to 25 wt%) and sustained in vitro release exceeding six months (larger particles that load more drug and last longer). These long-acting Feno-MP exhibited desired particle size suitable for intravitreal injection (IVT) through fine gauge needles. The lead Feno-MP-F6 maintained sustained drug levels in the retina for at least 6 months following a single intravitreal injection without obvious toxic issues in rats and rabbits. This study investigated a 6-month-long therapeutic effects of a single dose of fenofibrate-loaded microparticles (Feno-MP) via a non-VEGF, PPARα-dependent mechanism in streptozotocin (STZ)-induced diabetic rats (a DR model), Vldlr-/- mice (a wet-AMD model), and Abca4-/-/Rdh8-/- mice (a dry-AMD model). Comprehensive analyses revealed broad therapeutic benefits across all the three disease models. In the STZ-induced DR model, one single IVT dose of Feno-MP restored both photopic and scotopic electroretinogram (ERG) responses, decreased leukostasis, and enhanced blood-retinal barrier function over a period of 6 months. In Vldlr-/- mice, a wet-AMD model, Feno-MP effectively reduced both subretinal and intraretinal neovascularization and decreased retinal vascular leakage for at least 6 months. In Abca4-/-/Rdh8-/- mice, single Feno-MP treatment preserved photoreceptors, increased cone photoreceptors survival and improved mitochondrial function up to 6 months. Our study showed that Feno-MP may become a promising therapy for DR, wet-AMD and even dry-AMD with only 1-2 IVT injections/year. This work represents the approach to repurpose the oral drug fenofibrate for long-lasting intraocular delivery through a sustained release system.

Prior research has demonstrated the therapeutic potential of peroxisome proliferator-activated receptor α (PPARα) agonist fenofibrate on diabetic retinopathy. In the present study, a novel non-fibrate PPARα agonist, A190, was designed with higher potency and selectivity than fenofibrate in PPARα agonism. A190 was encapsulated in biodegradable microparticles (A190-MP) to ensure sustained drug release, with detection in the retina up to 6 months following a single intravitreal injection. A190-MP alleviated retinal dysfunction as shown by electroretinography in Vldlr-/- (wet-AMD model) and Abca4-/-/Rdh8-/- (dry-AMD model) mice. A190-MP also attenuated the decreases in cone photoreceptor density and outer nuclear layer thickness as demonstrated by optical coherence tomography and histology. Moreover, A190-MP reduced vascular leakage and neovascularization in Vldlr-/- mice, suggesting an anti-inflammatory and anti-angiogenic effect. A190-MP upregulated expression of PPARα, PGC1α, and TOMM20 in the retina of Vldlr-/- and Abca4-/-/Rdh8-/- mice. A190-MP also improved retinal mitochondrial function as shown by Seahorse analysis using retinal biopsy. In vitro, A190 attenuated oxidative stress and preserved cell viability in a photoreceptor-derived cell line exposed to 4-HNE and improved mitochondrial function, via a PPARα-dependent mechanism. These findings revealed sustained therapeutic effects of A190-MP in wet and dry AMD models, through improving mitochondrial function by activating PPARα.

To evade host antiviral response, viruses have evolved to take advantage of their noncoding RNAs (ncRNAs). Snakehead vesiculovirus (SHVV), a newly isolated fish rhabdovirus from diseased hybrid snakehead, has caused high mortality to the cultured snakehead fish during the past years in China. However, little is known about the mechanisms of its pathogenicity. Our study revealed that overexpression of the 30-nt leader RNA promoted SHVV replication. RNA-protein binding investigation revealed that SHVV leader RNA could interact with host 40S ribosomal protein S8 (RPS8) and 60S ribosomal protein L13a (L13a). Furthermore, we found that SHVV infection upregulated RPS8 and L13a, and in turn, overexpression of RPS8 or L13a inhibited, while knockdown of RPS8 or L13a promoted, SHVV replication, suggesting that RPS8 and L13a acted as host antiviral factors in response to SHVV infection. In addition, our study revealed that RPS8- or L13a-mediated inhibition of SHVV replication could be restored by co-transfection with leader RNA, suggesting that the interaction between leader RNA and RPS8 or L13a might affect the anti-SHVV effects of RPS8 and L13a. Taken together, these results suggest that SHVV leader RNA can interact with the host antiviral factors RPS8 and L13a, and promote SHVV replication. This study provides a better understanding of the molecular mechanism of the pathogenesis of SHVV and a potential antiviral strategy against SHVV infection.

Leader RNAs are viral small non-coding RNAs that has been proved to play important roles in viral replication. Snakehead vesiculovirus (SHVV) is an aquatic virus that has caused huge economic loss in Chinese snakehead fish aquaculture industry. It has been proved that SHVV would generate leader RNA during the process of infection, and leader RNA could interact with viral nucleoprotein to promote viral replication. In this study, we identified that leader RNA could also interact with cellular protein Cold Shock Domain containing E1 (CSDE1) and heterogeneous nuclear ribonucleoproteins A3 (hnRNP A3). Further investigation reveals that overexpression of CSDE1 and hnRNP A3 facilitated SHVV replication. Downregulation of CSDE1 and hnRNP A3 by siRNA inhibited SHVV replication. This study provided a new sight into understand the mechanism of SHVV replication, and a potential anti-SHVV target for drug research.

Diabetes can result in impaired corneal wound healing. Mitochondrial dysfunction plays an important role in diabetic complications. However, the regulation of mitochondria function in the diabetic cornea and its impacts on wound healing remain elusive. The present study aimed to explore the molecular basis for the disturbed mitochondrial metabolism and subsequent wound healing impairment in the diabetic cornea. Seahorse analysis showed that mitochondrial oxidative phosphorylation is a major source of ATP production in human corneal epithelial cells. Live corneal biopsy punches from type 1 and type 2 diabetic mouse models showed impaired mitochondrial functions, correlating with impaired corneal wound healing, compared to nondiabetic controls. To approach the molecular basis for the impaired mitochondrial function, we found that Peroxisome Proliferator-Activated Receptor-α (PPARα) expression was downregulated in diabetic human corneas. Even without diabetes, global PPARα knockout mice and corneal epithelium-specific PPARα conditional knockout mice showed disturbed mitochondrial function and delayed wound healing in the cornea, similar to that in diabetic corneas. In contrast, fenofibrate, a PPARα agonist, ameliorated mitochondrial dysfunction and enhanced wound healing in the corneas of diabetic mice. Similarly, corneal epithelium-specific PPARα transgenic overexpression improved mitochondrial function and enhanced wound healing in the cornea. Furthermore, PPARα agonist ameliorated the mitochondrial dysfunction in primary human corneal epithelial cells exposed to diabetic stressors, which was impeded by siRNA knockdown of PPARα , suggesting a PPARα-dependent mechanism. These findings suggest that downregulation of PPARα plays an important role in the impaired mitochondrial function in the corneal epithelium and delayed corneal wound healing in diabetes.