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Jude Al-Sabah, Valerie Stepps, Stephen Bartelmez, Maria Grant, Ashay Bhatwadekar; Inhibition of Transforming Growth Factor β1 (TGF-β1) Using Morpholino Antisense Promotes Repair in Diabetic CD34+ Cells by Modulating the Protein Ubiquitination Pathway. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3240.
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We identified a rare but highly significant cohort of diabetic patients who, in spite of having long-standing diabetes, did not show any evidence of diabetic retinopathy (DR). Gene array analysis of CD34+ stem cells from the protected population (when compared to age-, gender- and duration of diabetes-matched patients with DR) demonstrated downregulation of pathways mediated by TGF-β1. We further examined the potential role of TGF-β1 in patient CD34+ stem cells by transplanting these cells into mice with ischemia-reperfusion retinal damage. We showed that transient inhibition of TGF-β1 in diabetic CD34+ cells facilitated their reparative function in injured retinas. This study was undertaken to explore the mechanism responsible for the beneficial effect of TGF-β1 downregulation in CD34+ cells from a cohort of patients with DR.
Human CD34+ cells from the blood of healthy and diabetic individuals were isolated and treated with either control or TGF-β1 phosphorodiamidate morpholino oligomers (PMO). Cell samples were processed using Agilent Whole Human Genome Oligo microarrays. Data were analyzed using Ingenuity pathway analysis software.
An average of 741 molecules mapped canonical pathways in this study. Top functions were associated with protein synthesis, RNA post-transcriptional modifications, cell cycle regulation and cancer. The networks mapped in healthy and diabetic cells involved a variety of mRNA targets associated with TGF-β1 signaling, specifically RUNX2, SMURF1 and GRB2. Strikingly, TGF-β1 PMO treatment showed robust upregulation of the protein ubiquitination pathway in more than 80% of the patient population. About 33% of patients mapped protein ubiquitination as the top canonical pathway. Individual transcript analysis revealed that the balance between SnON (Ski-related novel protein N or SkiL), a newly identified repressor of TGF-β1, and Smurf 2 is critical for controlling downstream signaling of TGF-β1, which is mediated through Smad 4.
Our study identified protein ubiquitination as a key target after the inhibition of TGF-β1 (using PMO) in CD34+ cells. We propose that a balance between the protein levels of SkiL and Smurf 2 is critical to mediating the inhibitory effect of TGF-β1 PMO in diabetic CD34+ cells as a potential treatment for vasodegeneration in diabetic retinopathy.
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