June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Elevated expression of RUNX1 by vascular endothelial cells in proliferative diabetic retinopathy
Author Affiliations & Notes
  • Jonathan D. Lam
    Schepens Eye Research Institute, Boston, Massachusetts, United States
    Ophthalmology, Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Daniel Oh
    Schepens Eye Research Institute, Boston, Massachusetts, United States
    Ophthalmology, Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Patricia A D'Amore
    Schepens Eye Research Institute, Boston, Massachusetts, United States
    Ophthalmology, Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Leo A Kim
    Schepens Eye Research Institute, Boston, Massachusetts, United States
    Ophthalmology, Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Joseph Arboleda-Velasquez
    Schepens Eye Research Institute, Boston, Massachusetts, United States
    Ophthalmology, Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Jonathan Lam, None; Daniel Oh, None; Patricia D'Amore, 62/422,523 (P); Leo Kim, 62/422,523 (P); Joseph Arboleda-Velasquez, 62/422,523 (P)
  • Footnotes
    Support  NIH grants R01EY005318, R00EY021624, K12EY16335, and P30EY003790. ADA Innovation Award 7–12-IN-11, Massachusetts Lions Eye Research Fund, and the E. Matilda Ziegler Foundation for the Blind.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4249. doi:
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    • Get Citation

      Jonathan D. Lam, Daniel Oh, Patricia A D'Amore, Leo A Kim, Joseph Arboleda-Velasquez; Elevated expression of RUNX1 by vascular endothelial cells in proliferative diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4249.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : A major sight threatening complication of proliferative diabetic retinopathy (PDR) is the formation of fibrovascular membranes (FVMs). The pathophysiology of this process is poorly understood. Our whole transcriptomic analysis of CD31+ endothelial cells isolated from patient derived FVMs previously identified elevated transcripts of Runt-related transcription factor 1 (RUNX1), implicating it in PDR. RUNX1 has been studied extensively in hematopoietic development and various cancers but its role in endothelial cells is not fully elucidated. We test the hypothesis that RUNX1 plays an active role in endothelial cell glucose response and angiogenesis using in vitro tools.

Methods : Human microvascular retinal endothelial cells (HMREC) and human umbilical vein endothelial cells (HUVEC) were cultured in endothelial growth media supplemented with D-glucose or L-glucose as a control for osmolarity at concentrations of 5 mM (normal baseline) to 30 mM (high glucose) for 72 hours before analysis. qRT-PCR and Western blot with antibodies against RUNX1 and β-actin were performed. RUNX1 gene knockdown was achieved using small interfering RNA (siRNA) transfection using Dharmafect1 and migration and cell proliferation were studied using the scratch-wound assay and immunostaining for Ki67 respectively. Student’s t-test was used for statistical analysis.

Results : RNA expression of RUNX1 was elevated 2.8 ± 0.2 fold in HMREC cultured in high D-glucose (30 mM) compared to baseline controls (p<0.01, n=3). No difference in RNA expression of RUNX1 was seen in osmotic controls (L-glucose) (p>0.05). Western blotting analysis showed increased RUNX1 protein in both HMREC (1.28 ± 0.03, p<0.05, n=3) and HUVEC (1.3 ± 0.1, p<0.05, n=3) exposed to high glucose. Wound-closure in HMREC was significantly reduced by RUNX1 knockdown (0.052 ± 0.003 mm2/hr) compared to transfection control (0.131 ± 0.003 mm2/hr; p<0.001, n=5). RUNX1 knockdown in HMREC was also associated with decreased Ki67 staining (65% ± 4 of nuclei positively stained compared to 93% ± 2 in control; p<0.001, n=6).

Conclusions : These findings are consistent with the hypothesis that RUNX1 is inducible by hyperglycemia and contributes to a pro-angiogenic phenotype. Furthermore, these findings support the observation of elevated RUNX1 in endothelial cells from fibrovascular membranes of patients with PDR, suggesting a role for RUNX1 in pathologic retinal angiogenesis.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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