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![Inhibition of intravitreal neovascularization by AAV2-hPEDF treatment. (A) Representative micrographs of PAS-haematoxylin stained sections of the left (AAV2null-injected) and right (AAV2-hPEDF-injected) eyes from TgIGF-I mice 6 months after delivery of the vectors. Each pair of images corresponds to one animal. Note the presence of completely formed neovessels (insets, asterisks) in the vitreous cavity of AAV2-nullinjected eyes. Endothelial cells stain strongly for PAS (inset, arrows) [21]. Original magnification 206 (B) Endothelial cells on the vitreal side of the internal limiting membrane were counted in six non-consecutive sections per eye. AAV2-hPEDF treated eyes showed a striking reduction in the number of intravitreal PAS positive cells when compared with their untreated contralateral eyes. Two null-injected transgenic eyes could not be analysed due to marked retinal detachment that impeded the counting of PAS+ cells in the vitreous cavity. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; L, lenses; V, vitreous. doi:10.1371/journal.pone.0041511.g004](profile/Assumpcio-Bosch/publication/230715216/figure/fig10/AS:669644880891914@1536667189374/Inhibition-of-intravitreal-neovascularization-by-AAV2-hPEDF-treatment-A-Representative.jpg)
Inhibition of intravitreal neovascularization by AAV2-hPEDF treatment. (A) Representative micrographs of PAS-haematoxylin stained sections of the left (AAV2null-injected) and right (AAV2-hPEDF-injected) eyes from TgIGF-I mice 6 months after delivery of the vectors. Each pair of images corresponds to one animal. Note the presence of completely formed neovessels (insets, asterisks) in the vitreous cavity of AAV2-nullinjected eyes. Endothelial cells stain strongly for PAS (inset, arrows) [21]. Original magnification 206 (B) Endothelial cells on the vitreal side of the internal limiting membrane were counted in six non-consecutive sections per eye. AAV2-hPEDF treated eyes showed a striking reduction in the number of intravitreal PAS positive cells when compared with their untreated contralateral eyes. Two null-injected transgenic eyes could not be analysed due to marked retinal detachment that impeded the counting of PAS+ cells in the vitreous cavity. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; L, lenses; V, vitreous. doi:10.1371/journal.pone.0041511.g004
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Neovascularization associated with diabetic retinopathy (DR) and other ocular disorders is a leading cause of visual impairment and adult-onset blindness. Currently available treatments are merely palliative and offer temporary solutions. Here, we tested the efficacy of antiangiogenic gene transfer in an animal model that mimics the chronic progres...
Contexts in source publication
Context 1
... vascular struc- tures were detectable in the vitreous cavity of all transgenic eyes receiving AAV2-null empty vector ( Figure 3C), but no vessels were observed in the vitreous cavity of any of the contralateral eyes treated with AAV2-PEDF (data not shown). Similarly, after PAS- haematoxylin staining, no vessels were observed in the vitreous cavity of the right, PEDF-injected eyes ( Figure 4A), and the quantification of intravitreal PAS+ cells revealed that hPEDF gene delivery led to a striking reduction in the number of these cells, likely vascular cells [21], with respect to the untreated contralateral eye in all animals analyzed ( Figure 4B). Two null-injected transgenic eyes could not be analysed due to marked retinal detachment that impeded the counting of PAS+ cells in the vitreous cavity. ...
Context 2
... vascular struc- tures were detectable in the vitreous cavity of all transgenic eyes receiving AAV2-null empty vector ( Figure 3C), but no vessels were observed in the vitreous cavity of any of the contralateral eyes treated with AAV2-PEDF (data not shown). Similarly, after PAS- haematoxylin staining, no vessels were observed in the vitreous cavity of the right, PEDF-injected eyes ( Figure 4A), and the quantification of intravitreal PAS+ cells revealed that hPEDF gene delivery led to a striking reduction in the number of these cells, likely vascular cells [21], with respect to the untreated contralateral eye in all animals analyzed ( Figure 4B). Two null-injected transgenic eyes could not be analysed due to marked retinal detachment that impeded the counting of PAS+ cells in the vitreous cavity. ...
Context 3
... obtained from non-injected and AAV2-null injected TgIGF-I showed a 50% of increment in VEGF levels with respect to Wt ( Figure 7A). This increased VEGF in transgenic mice was normalized 6 months after a single treat- ment with AAV2-hPEDF ( Figure 7A), which was consistent with the inhibition of neovascularization observed ( Figures 4A, B, 5). ...
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Citations
... Until present, retinal explants are normally cultured with stimuli that portray the retinal environment in DR, such as high glucose, oxidative stress, or advanced glycation end-products (AGE) [196]. Other than that, the retina from the in vivo DR model was used for the ex vivo in vitro culture once the retinal neovascularisation development had taken place to represent the PDR process [197][198][199][200]. The use of retinal explants still needs to be improved in research, possibly due to the expertise requirements to isolate retinas and their unsuitability for long-term experimental periods [201]. ...
Diabetic retinopathy (DR), one of the leading causes of visual impairment and blindness worldwide, is one of the major microvascular complications in diabetes mellitus (DM). Globally, DR prevalence among DM patients is 25%, and 6% have vision-threatening problems among them. With the higher incidence of DM globally, more DR cases are expected to be seen in the future. In order to comprehend the pathophysiological mechanism of DR in humans and discover potential novel substances for the treatment of DR, investigations are typically conducted using various experimental models. Among the experimental models, in vivo models have contributed significantly to understanding DR pathogenesis. There are several types of in vivo models for DR research, which include chemical-induced, surgical-induced, diet-induced, and genetic models. Similarly, for the in vitro models, there are several cell types that are utilised in DR research, such as retinal endothelial cells, Müller cells, and glial cells. With the advancement of DR research, it is essential to have a comprehensive update on the various experimental models utilised to mimic DR environment. This review provides the update on the in vitro, in vivo, and ex vivo models used in DR research, focusing on their features, advantages, and limitations.
... Penelitian menggunakan PDEF dengan AAV2 sebagai vektor pada hewan coba menunjukkan turunnya kadar intraokular VEGF, yang dapat menghambat neovaskularisasi intravitreal, dan mencegah terjadinya ablasi retina. 60 Pendekatan terapi gen-spesifik dapat pula ditujukan untuk kepentingan proteksi retina, dengan cara mengintervensi degenerasi vaskular dan neuronal pada fase awal RD. RD ...
Retinopati Diabetik (RD) merupakan penyebab utama kebutaan pada usia produktif di seluruh dunia. Kontrol hiperglikemi dan tekanan darah yang buruk merupakan faktor risiko yang esensial dalam perkembangan RD. Kondisi ini akan memicu terjadinya mekanisme biomolekular yang menyebabkan kerusakan pada pembuluh dan sel di retina dan berkembang menjadi RD. Tahap awal RD umumnya tidak bergejala, sehingga direkomendasikan untuk melakukan penapisan RD segera setelah pasien terdiagnosis DM tipe 2, dan setiap 5 tahun bagi pasien DM tipe 1. Diagnosis RD didasarkan oleh temuan lesi retina yang diklasifikasikan menjadi RD non proliferatif dan proliferatif, dan edema makula diabetik. Terapi RD dapat dilakukan dengan pendekatan non-farmakologi dan farmakologi yang diterapkan berdasarkan derajat keparahan untuk meminimalisir progresivitas RD. Selain terapi konvensional, terdapat terapi potensial di masa depan, yaitu mencakup terapi farmakologi secara oral, terapi genetik, terapi sel punca, dan terapi CRISPR-Cas-Based.
... The implementation of gene therapy for DR, ideally as a one-time treatment, could be a long-term effective alternative to pharmacological strategies (Wang et al. 2020). However, respective approaches are still in the experimental stage by either targeting neovascularisation and vascular hyperpermeability or testing neuroprotective approaches in multiple DR-like animal models (Pechan et al. 2009, Haurigot et al. 2012, Zhang et al. 2015, Diaz-Lezama et al. 2016, Wang et al. 2020. ...
Diabetic retinopathy (DR) is considered a primarily microvascular complication of diabetes. Müller glia cells are at the center of the retinal neurovascular unit and play a critical role in DR. We therefore investigated Müller cell-specific signaling pathways that are altered in DR to identify novel targets for gene therapy. Using a multi-omics approach on purified Müller cells from diabetic db/db mice, we found the mRNA and protein expression of the glucocorticoid receptor (GR) to be significantly decreased, while its target gene cluster was down-regulated. Further, oPOSSUM TF analysis and ATAC- sequencing identified the GR as a master regulator of Müller cell gliosis in DR. Cortisol not only increased GR phosphorylation. It also induced changes in the expression of known GR target genes in retinal explants. Finally, retinal functionality was improved by AAV-mediated overexpression of GR in Müller cells. Our study demonstrates an important role of the glial GR in DR and implies that therapeutic approaches targeting this signalling pathway should be aimed at increasing GR expression rather than the addition of more ligand.
Graphical abstract
... In our previous study, RNA sequencing and bioinformatics analysis showed that DVDMS-SDT downregulates a number of genes including SERPINF1 (the mRNA encoding pigment epithelium-derived factor [PEDF]) and HIF-1A, which was blocked by caspase 3 specific inhibitor Ac-DEVD-CHO [18]. Previous studies have indicated that PEDF inhibits the expression and activities of MMP-2/9 in the aqueous humor of a proliferative diabetic retinopathy model [21] and in spontaneous pancreatic carcinoma [22]. However, the transcription factor HIF-1α promoted MMP-2/9 expression [23,24]. ...
... Therefore, we attributed the increase in the percentage content of collagen after the DVDMS-SDT treatment in this study to an inhibition of its degradation rather than an increase of its synthesis. Recently, we found that DVDMS-SDT, as a macrophage-specific targeted treatment, activates caspase 3 via the mitochondrial-apoptosis pathway to inhibit the expression of SER-PINF1 (the mRNA encoding PEDF) and HIF-1A [18], which are involved in the regulation of MMP-2 and MMP-9 [21][22][23][24]. In the present study, we found that DVDMS-SDT increased the expression of PEDF and decreased the expression of HIF-1 α by activating caspase 3 in macrophages, thereby inhibiting the expression of MMP-2 and MMP-9. ...
Background
The rupture of vulnerable atherosclerotic plaque is the main cause of acute ischemic vascular events, and is characterized by pathological degradation of matrix collagen in the fibrous cap. In a previous study, we reported that 5-aminolevulinic acid-mediated sonodynamic therapy suppressed collagen degradation in rabbit plaque. However, the underlying molecular mechanism has yet to be fully elucidated.
Methods
We applied sinoporphyrin sodium-mediated sonodynamic therapy (DVDMS-SDT) to balloon-denuded rabbit and apolipoprotein E-deficient (ApoE−/−) mouse models to observe collagen content in plaque. Cultured human THP-1 and mouse peritoneal macrophage-derived foam cells were used for in vitro mechanistic studies.
Results
We observed that DVDMS-SDT decreased plaque area and increased the percentages of collagen and smooth muscle cells and reduced the percentage of macrophages in rabbit and ApoE-/- mouse advanced plaques. In vitro , DVDMS-SDT modulated the caspase 3-pigment epithelium-derived factor/hypoxia-inducible factor-1α (PEDF/HIF-1α)-matrix metalloprotease-2/9 (MMP-2/MMP-9) signaling in macrophage foam cells.
Conclusions
Our findings show that DVDMS-SDT effectively inhibits matrix collagen degradation in advanced atherosclerotic plaque by modulating caspase 3-PEDF/HIF-1α-MMP-2/MMP-9 signaling in macrophage foam cells and therefore represents a suitable and promising clinical regimen to stabilize vulnerable plaques.
... PEDF is a multifunctional protein with critical roles in many pathological processes. Researchers found that PEDF is related to angiogenesis and PEDF-modified PDR mice presented with decreased VEGF expression, inflammation and neovascularization level, offering evidence for clinical use of PEDF modification to treat PDR [104,105]. CAD is another antiangiogenic factor selected as genetic target for PDR therapy. Intravitreally AAV-delivered CAD can significantly reduce both choroidal and retinal neovascularization in a laser-induced and OIR model [106]. ...
Diabetic retinopathy is the major blinding disease among working-age populations, which is becoming more significant due to the growth of diabetes. The metabolic-induced oxidative and inflammatory stress leads to the insult of neovascular unit, resulting in the core pathophysiology of diabetic retinopathy. Existing therapies focus on the inflammation, oxidation, and angiogenesis phenomena of diabetic retinopathy, without effect to radically cure the disease. This review also summarizes novel therapeutic attempts for diabetic retinopathy along with their advantages and disadvantages, mainly focusing on those using cellular and genetic techniques to achieve remission on a fundamental level of disease.
... Among the viable drug delivery routes utilized to suppress VEGF-mediated angiogenesis, lipidic nanocarriers have been engineered to silence the expression of the human antigen R (HuR/Elavl1), encoding an RNA-binding protein known to be involved in diabetic retinopathy, suggesting a similar approach could be leveraged for the treatment of wet age-related macular degeneration and diabetic macular oedema [255]. Ocular angiogenesis has also been tackled by mimicking pharmacologic VEGF inhibition through lentiviral and adenoviral delivery of siRNA and miRNA, or by inducing the expression of the anti-angiogenic PEDF [256,257]. ...
Retinal neurogenesis is driven by concerted actions of transcription factors, some of which are expressed in a continuum and across several cell subtypes throughout development. While seemingly redundant, many factors diversify their regulatory outcome on gene expression, by coordinating variations in chromatin landscapes to drive divergent retinal specification programs. Recent studies have furthered the understanding of the epigenetic contribution to the progression of age-related macular degeneration, a leading cause of blindness in the elderly. The knowledge of the epigenomic mechanisms that control the acquisition and stabilization of retinal cell fates and are evoked upon damage, holds the potential for the treatment of retinal degeneration. Herein, this review presents the state-of-the-art approaches to investigate the retinal epigenome during development, disease, and reprogramming. A pipeline is then reviewed to functionally interrogate the epigenetic and transcriptional networks underlying cell fate specification, relying on a truly unbiased screening of open chromatin states. The related work proposes an inferential model to identify gene regulatory networks, features the first footprinting analysis and the first tentative, systematic query of candidate pioneer factors in the retina ever conducted in any model organism, leading to the identification of previously uncharacterized master regulators of retinal cell identity, such as the nuclear factor I, NFI. This pipeline is virtually applicable to the study of genetic programs and candidate pioneer factors in any developmental context. Finally, challenges and limitations intrinsic to the current next-generation sequencing techniques are discussed, as well as recent advances in super-resolution imaging, enabling spatio-temporal resolution of the genome.
... The upregulation of PEDF and IGF-1 we identified in RPE-µ Ts has important therapeutic implications, as both molecules provide potent photoreceptor pro-survival signals [67][68][69][70][71][72][73][74][75] and have been shown to rescue photoreceptors in retinal degeneration animal models [12,70,74]. Enriched photoreceptors have been reported to rapidly degenerate in vitro, partially due to the lack of support from RPE [76,77]. ...
... We hypothesize that PEDF and IGF-1 are likely at least partially responsible, although this prediction would require further experimentation in a more sophisticated model of retinal degeneration. Increased secretion of PEDF in RPE-µ Ts is of particular interest, as this neurotrophic factor has been shown to have clear therapeutic potential in the treatment of retinal degeneration by preventing photoreceptor apoptosis [67,68,78,79]. ...
The retinal pigmented epithelium (RPE) plays a critical role in photoreceptor survival and function. RPE deficits are implicated in a wide range of diseases that result in vision loss, including age-related macular degeneration (AMD) and Stargardt disease, affecting millions worldwide. Subretinal delivery of RPE cells is considered a promising avenue for treatment, and encouraging results from animal trials have supported recent progression into the clinic. However, the limited survival and engraftment of transplanted RPE cells delivered as a suspension continues to be a major challenge. While RPE delivery as epithelial sheets exhibits improved outcomes, this comes at the price of increased complexity at both the production and transplant stages. In order to combine the benefits of both approaches, we have developed size-controlled, scaffold-free RPE microtissues (RPE-µTs) that are suitable for scalable production and delivery via injection. RPE-µTs retain key RPE molecular markers, and interestingly, in comparison to conventional monolayer cultures, they show significant increases in the transcription and secretion of pigment-epithelium-derived factor (PEDF), which is a key trophic factor known to enhance the survival and function of photoreceptors. Furthermore, these microtissues readily spread in vitro on a substrate analogous to Bruch’s membrane, suggesting that RPE-µTs may collapse into a sheet upon transplantation. We anticipate that this approach may provide an alternative cell delivery system to improve the survival and integration of RPE transplants, while also retaining the benefits of low complexity in production and delivery.
... Furthermore, eye drop delivery of PEDF-34 promoted ganglion cell survival and axon regeneration after optic nerve crush injury in rats [92]. Gene therapy using AAV-mediated PEDF [93] or the engineered episomal vector pEPito (pEPito-PEDF) [94,95] has also exhibited promising results in experimental in vivo models. ...
The concept of diabetic retinopathy as a microvascular disease has evolved and is now considered a more complex diabetic complication in which neurovascular unit impairment plays an essential role and, therefore, can be considered as a main therapeutic target in the early stages of the disease. However, neurodegeneration is not always the apparent primary event in the natural story of diabetic retinopathy, and a phenotyping characterization is recommendable to identify those patients in whom neuroprotective treatment might be of benefit. In recent years, a myriad of treatments based on neuroprotection have been tested in experimental models, but more interestingly, there are drugs with a dual activity (neuroprotective and vasculotropic). In this review, the recent evidence concerning the therapeutic approaches targeting neurovascular unit impairment will be presented, along with a critical review of the scientific gaps and problems which remain to be overcome before our knowledge can be transferred to clinical practice.
... Our data further substantiate the notion that VEGF level is not altered by the RRD or its severity. PEDF exhibits anti-angiogenic effects 25 and is essential for neuronal development in the retina 26 , and supplementation with PEDF has a significant neuroprotective effect 27 . Recently, Takahashi et al. reported that vitreous levels of IL-6, IL-8, and IP-10 were elevated in RRD eyes 24 , and IL-8 was significantly correlated with the extent of the detached area. ...
Many studies have demonstrated that rhegmatogenous retinal detachment (RRD) leads to impaired retinal circulation. However, the involvement of inflammation in the RRD-induced worsening of retinal circulation was obscure. This retrospective observational study included 150 patients with primary RRD (macula-on, n = 63; macula-off, n = 87) who underwent 25-gauge microincision vitrectomy surgery (25G MIVS). Total retinal blood flow was represented by the mean blur rate (MBR) of the optic nerve head vessel, measured by laser speckle flowgraphy preoperatively and until 6 months postoperatively. Aqueous humor samples were obtained during surgery to determine cytokine concentrations by enzyme-linked immunosorbent assay. At 3 and 6 months postoperatively, there were no significant differences between eyes with macula-on RRD and fellow eyes. However, in macula-off RRD, MBR remained significantly lower in RRD eyes 6 months postoperatively (P < 0.05). Log-transformed levels of soluble intercellular adhesion molecule-1 (sICAM-1) were negatively correlated with relative MBR (r-MBR, RRD eye/fellow eye) before surgery (r = − 0.47, P = 0.01) in macula-on, but not macula-off, RRD. Six months postoperatively, r-MBR correlated significantly with sICAM-1 levels (r = − 0.36, P = 0.02) in macula-off RRD. ICAM-1 may play a role in RRD-induced deterioration of retinal circulation.
... A unique specific band was detected at 50 kDa, compared with the molecular weight, as was predicted for the PEDF protein. In addition, this antibody has been extensively used on western blotting and immunofluorescence staining, found in previous published experiments carried out in mouse tissue (Haurigot et al., 2012;Hou et al., 2010). ...
Pigment epithelium‐derived factor (PEDF) is a multifunctional protein which was initially described in the retina, although it is also present in other tissues. It functions as an antioxidant agent promoting neuronal survival. Recently, a PEDF receptor has shown an elevated binding affinity for PEDF. There are no relevant data regarding the distribution of both proteins in the brain, therefore the main goal of this work was to investigate the spatiotemporal presence of PEDF and PEDFR in the adult mouse brain, and to determine the PEDF blood level in mouse and human. The localization of both proteins was analyzed by different experimental methods such as immunohistochemistry, western‐blotting, and also by enzyme‐linked immunosorbent assay. Differential expression was found in some telencephalic structures and positive signals for both proteins were detected in the cerebellum. The magnitude of the PEDFR labeling pattern was higher than PEDF and included some cortical and subventricular areas. Age‐dependent changes in intensity of both protein immunoreactions were found in the cortical and hippocampal areas with greater reactivity between 4 and 8 months of age, whilst others, like the subventricular zones, these differences were more evident for PEDFR. Although ubiquitous presence was not found in the brain for these two proteins, their relevant functions must not be underestimated. It has been described that PEDF plays an important role in neuroprotection and data provided in the present work represents the first extensive study to understand the relevance of these two proteins in specific brain areas.
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