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Dolly Ann Padovani-Claudio, Yueli Zhang, John S. Penn; CXCL8 can increase retinal microvascular endothelial cell (hRMEC) migration rate and its receptor signals may be linked to VEGF-induced migration.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2509. doi: https://doi.org/.
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Increased interleukin 8 (IL-8/CXCL8) and VEGF levels in the vitreous correlate with diabetic retinopathy (DR) progression and angiogenesis. We have previously reported CXCL8 induction by diabetes-relevant stimuli in human retinal microvascular endothelial (hRMEC) and Müller cells. There is evidence in the cancer literature that CXCL8 and VEGF angiogenic signals may be linked, but this has not been investigated in the context of DR. We aim to compare CXCL8 and VEGF’s effects on hRMEC migration and the potential of CXCL8 receptor inhibitors to reduce their effects.
hRMECs were grown in attachment factor coated 12-well plates and stimulated with vehicle, 25ng/mL VEGF, or 10ng/mL human recombinant CXCL8, in the presence or absence of 30min pre-treatment with 50ng/mL of Reparixin (a CXCL8 receptor inhibitor). A magnetic stencil migration assay was used to quantify migration at 0, 6, 24, 33 and 48hr post-stimulation. Data was recorded by a blind observer and plotted as percentage of the area devoid of cells at time zero filled at each time-point. Two-way ANOVA was used to determine statistical significance (P<0.001**; N=6 per group).
At 6hr, migration was not significantly different between the groups. At 24hrs, the filled area in VEGF- and CXCL8-stimulated cultures was 20%** and 23%** larger than vehicle-treated cultures, respectively. By 33hrs, the mean increase in filled area compared to vehicle was 31%** for both. Reparixin pre-treatment significantly reduced VEGF- or CXCL8-induced migration by 16%** at 24hr and by 22%** at 33hrs, to levels similar to vehicle-treated cultures. There was no significant difference in filled area between vehicle- and Reparixin-treated cells at any time point. No cell morphology change was evident in the cultures.
CXCL8, which is highly upregulated in the vitreous of patients with DR, increased the rate of hRMEC migration, while the CXCL8 receptor inhibitor, Reparixin, limited hRMEC migration induction by both CXCL8 and VEGF independently. This suggests that signals from CXCL8 and VEGF receptors may be linked in hRMEC, as has been postulated for tumor and other endothelial cells. These findings support the notion that targeting the CXCL8 signaling pathway may be beneficial in DR and other pro-angiogenic retinal diseases.
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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