Development of strategies to modulate gene expression of angiogenesis-related molecules in the retina.

Intravitreal anti-vascular endothelial growth factor agents are the gold standard treatment of ocular neovascular diseases. However, their short-term efficacy implies frequent intravitreal injections. Gene therapy has the ability to provide longer duration of the therapeutic effect. We have previously described the effectiveness of the self-replicating episomal vector, pEPito, in long-term gene expression in mouse retina. In this study, we evaluated different constructs to overexpress pigment epithelium-derived factor (PEDF), an angiogenesis inhibitor, and simultaneously, to silence placental growth factor (PlGF), a key player in neovascularization. We employed the human cytomegalovirus promoter to drive the expression of PEDF and PlGF shRNA, in conjunction with cis-acting ribozymes, using pEPito as expressing vector. Our results demonstrated that the non-viral systems were able to efficiently promote a sustained increase of the PEDF: PlGF ratio in the mice retina, decreased in pathological conditions. This innovative approach could open avenues for the development of new therapeutic strategies.

Dual-Acting Antiangiogenic Gene Therapy Reduces Inflammation and Regresses Neovascularization in Diabetic Mouse Retina.

Intravitreal injections of anti-vascular endothelial growth factor drugs have become the gold standard treatment for diabetic retinopathy (DR). However, several patients are classified as non-responders or poor responders to treatment. Therefore, it is essential to study alternative target molecules. We have previously shown that the progression of DR in the Ins2Akita mouse reflects the imbalance between pro- and anti-angiogenic molecules found in the human retina. We report, for the first time, the therapeutic potential of a dual-acting antiangiogenic non-viral gene therapy. We have used an expressing vector encoding both the pigment epithelium-derived factor gene and a short hairpin RNA (shRNA) targeted to the placental growth factor to restore the balance between these factors in the retina. Twenty-one days after a single subretinal injection, we observed a marked decrease in the inflammatory response in the neural retina and in the retinal pigment epithelium, together with reduced vascular retinal permeability in the treated diabetic mouse. These results were accompanied by the restoration of the retinal capillary network and regression of neovascularization, with significant improvement of DR hallmarks. Concomitant with the favorable therapeutic effects, this approach did not affect retinal ganglion cells. Hence our results provide evidence toward the use of this approach in DR treatment.

PlGF silencing combined with PEDF overexpression: Modeling RPE secretion as potential therapy for retinal neovascularization.

Ocular neovascularization is a defining feature of several blinding diseases. We have previously described the effectiveness of long-term pigment epithelium-derived factor (PEDF) expression in the retina of diabetic mice in ameliorating some diabetic retinopathy hallmarks. In this study, we aimed to investigate if the antiangiogenic potential of PEDF overexpression was enhanced in combination with placental growth factor (PlGF) silencing. Human RPE cells were transfected with a self-replicating episomal vector (pEPito) for PEDF overexpression and/or a siRNA targeting PlGF gene. Conditioned media from PEDF overexpression, from PlGF inhibition and from their combination thereof were used to culture human umbilical vein endothelial cells, and their proliferation rate, migration capacity, apoptosis and ability to form tube-like structures were analyzed in vitro. We here demonstrate that pEPito-driven PEDF overexpression in combination with PlGF silencing in RPE cells does not affect their viability and results in an enhanced antiangiogenic activity in vitro. We observed a significant decrease in the migration and proliferation of endothelial cells, and an increase in apoptosis induction as well as a significant inhibitory effect on tube formation. Our findings demonstrate that simultaneous PEDF overexpression and PlGF silencing strongly impairs angiogenesis compared with the single approaches, providing a rationale for combining these therapies as a new treatment for retinal neovascularization.

Dysregulation of trophic factors contributes to diabetic retinopathy in the Ins2Akita mouse.

Diabetic retinopathy (DR) is considered as a diabetes-related complication that can lead to severe visual impairments. By 2030, it is expected that 1 in 5 adults will suffer from the disease. Suitable animal models for chronic DR are essential for a better understanding of the pathophysiology and to further develop new treatments. The Ins2Akita mouse is a type 1 diabetes model that shows signs of both early and late stages of DR, including pericyte loss, increased vascular permeability, increased acellular capillaries and neovascularization. To further characterize DR in the Ins2Akita mouse model, we have evaluated the protein levels of the angiogenesis inducers vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) and the angiogenesis inhibitor pigment epithelium-derived factor (PEDF). Additionally, we have analyzed the protein expression profile of the glial markers ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) as well as of the chemokine monocyte chemoattractant protein 1 (MCP-1). In this study we demonstrate that, with disease progression, there is the development of an inflammatory response and an unbalanced expression of pro- and antiangiogenic factors in the neural retina and in the retinal pigment epithelium (RPE) of Ins2Akita mice. Therefore, our data provide support for the diabetic retinopathy features detected in the Ins2Akita retina, reflecting what is observed in the human pathology.

The role of the retinal pigment epithelium and Müller cells secretome in neovascular retinal pathologies.

Secreted trophic factors are key to maintain the structural and functional integrity of the retina, as they regulate cellular pathways responsible for survival, function, and response to injury. Nevertheless, these same factors can also be involved in retinal pathologies, as a consequence of the impairment of the secretory function of cells. The cells considered as major contributors to the retinal secretome are the retinal pigmented epithelium (RPE) and Müller cells. Their role in the pathophysiology of the most common neovascular pathologies in the retina - Age-related Macular Degeneration (AMD), Diabetic Retinopathy (DR), and Retinopathy of Prematurity (ROP) - is highlighted in this short review, together with current trophic factor-based therapies, which are mainly focused on controlling inflammation, cell survival, and angiogenesis.