Retinal Neuroprotective Effect of Mesenchymal Stem Cells Secretome Through Modulation of Oxidative Stress, Autophagy, and Programmed Cell Death.

Purpose: Degenerative mechanisms of retinal neurodegenerative diseases (RND) share common cellular and molecular signalization pathways. Curative treatment does not exist and cell-based therapy, through the paracrine properties of mesenchymal stem cells (MSC), is a potential unspecific treatment for RND. This study aimed to evaluate the neuroprotective capability of human bone marrow (bm) MSC secretome and its potential to modulate retinal responses to neurodegeneration. Methods: An in vitro model of spontaneous retinal neurodegeneration was used to compare three days of monocultured neuroretina (NR), NR cocultured with bmMSC, and NR cultured with bmMSC secretome. We evaluated retinal morphology markers (Lectin peanut agglutinin, rhodopsin, protein kinase C α isoform, neuronal-specific nuclear protein, glial fibrillary acidic protein, TdT-mediated dUTP nick-end labeling, and vimentin) and proteins involved in apoptosis (apoptosis-inductor factor, caspase-3), necroptosis (MLKL), and autophagy (p62). Besides, we analyzed the relative mRNA expression through qPCR of genes involved in apoptosis (BAX, BCL2, CASP3, CASP8, CASP9), necroptosis (MLKL, RIPK1, RIPK3), autophagy (ATG7, BCLIN1, LC3B, mTOR, SQSTM1), oxidative stress (COX2, CYBA, CYBB, GPX6, SOD1, TXN2, TXNRD1) and inflammation (IL1, IL6, IL10, TGFb1, TNFa). Results: The bmMSC secretome preserves retinal morphology, limits pro-apoptotic- and pro-necroptotic-related gene and protein expression, modulates autophagy-related genes and proteins, and stimulates the activation of antioxidant-associated genes. Conclusions: The neuroprotective ability of the bmMSC secretome is associated with activation of antioxidant machinery, modulation of autophagy, and inhibition of apoptosis and necroptosis during retinal degeneration. The neuroprotective effect of bmMSC secretomes in the presence/absence of MSC looks similar. Our current results reinforce the hypothesis that the human bmMSC secretome slows retinal neurodegeneration and may be a therapeutic option for treating RND.

Programmed Cell Death and Autophagy in an in vitro Model of Spontaneous Neuroretinal Degeneration.

Retinal neurodegenerative diseases are the leading causes of visual impairment and irreversible blindness worldwide. Although the retinal response to injury remains closely similar between different retinal neurodegenerative diseases, available therapeutic alternatives are only palliative, too expensive, or very specific, such as gene therapy. In that sense, the development of broad-spectrum neuroprotective therapies seems to be an excellent option. In this regard, it is essential to identify molecular targets involved in retinal degeneration, such as cell death mechanisms. Apoptosis has been considered as the primary cell death mechanism during retinal degeneration; however, recent studies have demonstrated that the only use of anti-apoptotic drugs is not enough to confer good neuroprotection in terms of cell viability and preservation. For that reason, the interrelationship that exists between apoptosis and other cell death mechanisms needs to be characterized deeply to design future therapeutic options that simultaneously block the main cell death pathways. In that sense, the study aimed to characterize the programmed cell death (in terms of apoptosis and necroptosis) and autophagy response and modulation in retinal neurodegenerative diseases, using an in vitro model of spontaneous retinal neurodegeneration. For that purpose, we measured the mRNA relative expression through qPCR of a selected pool of genes involved in apoptosis (BAX, BCL2, CASP3, CASP8, and CASP9), necroptosis (MLKL, RIPK1, and RIPK3), and autophagy (ATG7, BCLIN1, LC3B, mTOR, and SQSTM1); besides, the immunoexpression of their encoding proteins (Casp3, MLKL, RIPK1, LC3B, and p62) were analyzed using immunohistochemistry. Our results showed an increase of pro-apoptotic and pro-necroptotic related genes and proteins during in vitro retinal neurodegeneration. Besides, we describe for the first time the modulation between programmed cell death mechanisms and autophagy in an in vitro retinal neurodegeneration model. This study reinforces the idea that cell death mechanisms are closely interconnected and provides new information about molecular signaling and autophagy along the retinal degeneration process.