Figure 7: VEGF ameliorates sphingolipid imbalance in NP–C iPSC neurons.
From: Pathological roles of the VEGF/SphK pathway in Niemann–Pick type C neurons
(a) Left, normal, hNPC-3 and hNPC-17 iPSCs generated nestin-positive neuroprogenitor cells (scale bar, 50 μm). Right, representative images of immunocytochemical staining the β-III tubulin following neural differentiation (scale bar, 50 μm). (b) hNPC-3 and hNPC-17 neurons were treated with human BM-MSCs, VEGFtg BM-MSCs or recombinant VEGF (10 ng ml−1). Three days after treatment, VEGF levels were measured in cell lysates. (c–f) SphK activity (c), sphingosine (d), S1P (e) and sphingomyelin (f) were measured in normal iPSC neurons and hNPC neurons with or without treatment. (g) Filipin staining of unesterified cholesterol in hNPC-3 neurons with or without treatment of recombinant VEGF for 3 days (scale bar, 50 μm). Quantification of filipin fluorescence intensities normalized to normal neurons. Unesterified cholesterol levels in normal iPSC neurons and hNPC neurons with or without treatment were measured (n=6 per group). (h) Effect of the VEGFR2 inhibitor on VEGF mediated sphingolipid modulation. hNPC-3 neurons were pretreated with PTK787 at 10 μM for 1 day and were treated for 3 days with 10 ng ml−1 VEGF and then assayed for SphK activity, sphingosine and S1P (n=7 per group). (i,j) Effect of NPC1 knockdown on sphingolipid factors in normal iPSC neurons. (i) Three days after NPC1 siRNA transfection, we measured the levels of NP C1 mRNA and VEGF expression. (j) SphK activity, sphingosine and S1P were measured in normal iPSC neurons treated with control or NPC1 siRNA (control, n=7; NPC1 siRNA, n=9). (k) Effect of VEGF knockdown on sphingolipid factors in normal iPSC neurons. Three days after VEGF siRNA transfection, we measured the levels of VEGF mRNA, SphK activity, sphingosine and S1P in normal iPSC neurons (n=7 per group). (l) Effect of a specific SphK1 inhibitor on sphingolipid factors in normal iPSC neurons. Normal neurons were treated with or without 20 μM SK1-I for 6 h. Lipids were extracted and sphingosine, S1P and unesterified cholesterol levels were determined (n=6 per group). b–h, one-way analysis of variance, Tukey’s post hoc test. i–l, Student’s t-test. *P<0.05, **P<0.01, ***P<0.005. All error bars indicate s.e.m.
