Abstract
Bone marrow-derived cells were demonstrated to improve organ function, but the lack of cell retention within injured organs suggests that the protective effects are due to factors released by the cells. Herein, we tested cell therapy using early outgrowth cells (EOCs) or their conditioned media (CM) to protect the retina of diabetic animal models (type 1 and type 2) and assessed the mechanisms by in vitro study. Control and diabetic (db/db) mice (8 weeks of age) were randomized to receive a unique intravenous injection of 5×105EOCs or 0.25 ml thrice weekly tail-vein injections of 10x concentrated CM and Wystar Kyoto rats rendered diabetic were randomized to receive 0.50 ml thrice weekly tail-vein injections of 10x concentrated CM. Four weeks later, the animals were euthanized and the eyes were enucleated. Rat retinal Müller cells (rMCs) were exposed for 24 h to high glucose (HG), combined or not with EOC-conditioned medium (EOC-CM) from db/m EOC cultures. Diabetic animals showed increase in diabetic retinopathy (DR) and oxidative damage markers; the treatment with EOCs or CM infusions significantly reduced this damage and re-established the retinal function. In rMCs exposed to diabetic milieu conditions (HG), the presence of EOC-CM reduced reactive oxygen species production by modulating the NADPH-oxidase 4 system, thus upregulating SIRT1 activity and deacetylating Lys-310-p65-NFκB, decreasing GFAP and VEGF expressions. The antioxidant capacity of EOC-CM led to the prevention of carbonylation and nitrosylation posttranslational modifications on the SIRT1 molecule, preserving its activity. The pivotal role of SIRT1 on the mode of action of EOCs or their CM was also demonstrated on diabetic retina. These findings suggest that EOCs are effective as a form of systemic delivery for preventing the early molecular markers of DR and its conditioned medium is equally protective revealing a novel possibility for cell-free therapy for the treatment of DR.
Highlights
With the incidence of diabetes increasing at an alarming rate, the number of people with diabetic retinopathy (DR) is expected to grow from 126.6 million to 191 million by 2030 [1]
Our results indicate that the intravenous infusion of early out growth cells (EOCs) or their conditioned media (CM) mitigates the early DR markers, namely glial fibrillary acidic protein (GFAP) and VEGF, and oxidative markers in diabetic retinas of db/db mice and fully restored retinal function in diabetic Wystar Kyoto rat (WKY) rats
Cell therapy using EOCs or using the CM either from db/m or from isogenic rat WKY cultured EOCs were shown to be potential therapies preventing of the early changes in DR
Summary
With the incidence of diabetes increasing at an alarming rate, the number of people with diabetic retinopathy (DR) is expected to grow from 126.6 million to 191 million by 2030 [1]. The known pathways, including oxidative stress, increased formation of AGEs, and activation of protein kinase-C and hexosamine pathways [2,3], appear to cross-talk. Mass spectrometric analysis of the EOC-conditioned medium (EOC-CM) identified proteins that regulate cellular functions implicated in fibrosis and proteins involved in stress responses, such as HSP-19, glutathione S transferase (GST1), peroxiredoxin, superoxide dismutase (SOD), thioredoxin, and heme oxygenase-1 [15]. These results indicate that EOCs display a great capacity for secreting soluble factor(s) with potent antioxidant activity that, when injected intravenously, replicate the salutary effects of the cells themselves
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