June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Inhibition of Transforming Growth Factor β1 (TGF-β1) Using Morpholino Antisense Promotes Repair in Diabetic CD34+ Cells by Modulating the Protein Ubiquitination Pathway
Author Affiliations & Notes
  • Jude Al-Sabah
    Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL
  • Valerie Stepps
    Beta Stem Therapeutics, San Francisco, CA
  • Stephen Bartelmez
    Beta Stem Therapeutics, San Francisco, CA
  • Maria Grant
    Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL
  • Ashay Bhatwadekar
    Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL
  • Footnotes
    Commercial Relationships Jude Al-Sabah, None; Valerie Stepps, None; Stephen Bartelmez, BetaStem Therapeutics (P); Maria Grant, None; Ashay Bhatwadekar, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3240. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 499 diabetic retinopathy • 498 diabetes  
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