Conditioned Medium from Early-Outgrowth Bone Marrow Cells Is Retinal Protective in Experimental Model of Diabetes

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.

Defective Autophagy in Diabetic Retinopathy

“…Immunoprecipitation was performed as before. 24 A total of 500 lg of protein diluted in extraction buffer were immunoprecipitated with goat anti-caspase 8 (Santa Cruz Biotechnology) using protein A/G agarose beads. The samples underwent Western blotting against p62/SQTSM1 (1:1000, Abcam, Cambridge, UK) followed by the appropriate secondary antibody.…”

Deficient Autophagy Contributes to the Development of Diabetic Retinopathy

“…Multiple mechanisms are triggered under hyperglycemic conditions (hexosamine and polyol pathways [41], synthesis de novo of diacylglycerol-PKC [42,43], low grade oxidative stress [44][45][46], inflammation [47][48][49][50][51], and advanced glycation end products [52,53]). Although vascular changes are presumed to be the hallmarks of DR, abnormalities in retinal function are detected in patients with diabetes who have good visual acuity [54][55][56][57][58][59].…”

“…The characteristics of retinal neurodegeneration are apoptosis of neuro cells and dysfunction of glial cells-mainly Müller cells [29,50,60]. In microvascular disease of diabetic retinopathy, both inner and outer blood retinal barrier break down [61].…”

δ Opioid Receptor Agonism Preserves the Retinal Pigmented Epithelial Cell Tight Junctions and Ameliorates the Retinopathy in Experimental Diabetes

“…Retinal function was measured using the UTAS-E3000 system (LKC Technologies, Inc., Gaithersburg, MD, USA), as previously described. [24][25][26][27] The measurements were taken after overnight dark adaptation. An intensity-response was recorded using a ganzfeld flash with an intensity of 0.25 cd.s/m 2 at 0 to 250 or 0 to 4000 milliseconds.…”

“…The immunohistochemistry was performed as previously described. 24 The retinal sections were incubated with anti-GFAP and anti-VEGF (1:250, Santa Cruz Biotechnology) overnight at 48C. The slides were then incubated with the appropriate secondary antibodies.…”