Increase in acid sphingomyelinase level in human retinal endothelial cells and CD34+ circulating angiogenic cells isolated from diabetic individuals is.

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Increase in acid sphingomyelinase level in human retinal endothelial cells and CD34+ circulating angiogenic cells isolated from diabetic individuals is associated with dysfunctional retinal vasculature and vascular repair process in diabetes Nermin Kady, Yuanqing Yan, PhD, Tatiana Salazar, PhD, Qi Wang, PhD, Harshini Chakravarthy, PhD, Chao Huang, Eleni Beli, PhD, Svetlana Navitskaya, Maria Grant, MD, Julia Busik, PhD Journal of Clinical Lipidology Volume 11, Issue 3, Pages 694-703 (May 2017) DOI: 10.1016/j.jacl.2017.03.007 Copyright © 2017 National Lipid Association Terms and Conditions

Figure 1 Diabetes induced increase in ASM expression. Total RNA was isolated and the transcript level of ASM was analyzed by quantitative real-time polymerase chain reaction in (A) HRECs, (B) CD34+ cells, and (C) plasma from diabetic donors (n = 4–7) compared with control donors (n = 3–4). Data are means ± standard error of the mean. *P < .05, significantly different as determined by Student's t-test. ASM, acid sphingomyelinase; HRECs, human retinal endothelial cells. Journal of Clinical Lipidology 2017 11, 694-703DOI: (10.1016/j.jacl.2017.03.007) Copyright © 2017 National Lipid Association Terms and Conditions

Figure 2 Diabetes impairs tube formation capacity of HRECs. (A) Postmortem imaging of human retina. Control retina with well-organized blood vessels (left), diabetic retina with signs of NPDR, intraretinal hemorrhages, and microaneurysms (right). (B) An in vitro tube formation assay was performed in control (n = 4) and diabetic (n = 7) HRECs using Matrigel matrix 96-well plate. Representative images of tube-like structures are shown. The cells were stained with Qtracker 525 (green), images were taken in ×10 magnification and total tube areas were calculated using MetaMorph software system. Quantification of tube area is shown on far right. Data are means ± standard error of the mean. *P < .05, significantly different as determined by Student's t-test. Scale bar = 50 μm. HRECs, human retinal endothelial cells; NPDR, nonproliferative diabetic retinopathy. Journal of Clinical Lipidology 2017 11, 694-703DOI: (10.1016/j.jacl.2017.03.007) Copyright © 2017 National Lipid Association Terms and Conditions

Figure 3 Reduced incorporation of diabetic CD34+ CACs into diabetic HRECs tubes. Tube formation by HRECs (Qtracker 525, green) isolated from control (A) or diabetic (B) donors either without CACs (left panel), or co-incubated with control (middle panel) or diabetic (right panel) CACs (Qtracker 655, red) is shown. Quantification of percentage of CD34+ CACs incorporation into HRECs tubes is shown on far right. Data are means ± standard error of the mean (n = 4–7). ***P < .0001, significantly different from control as determined by Student's t-test; not significant at P > .05. Scale bar = 50 μm. (C) CD34+ CACs alone were not able to form tube-like structures (left panel), but incorporated into HREC tubes, forming tube-like structures, when co-cultured with HRECs (right 3 panels). CACs, circulating angiogenic cells; HRECs, human retinal endothelial cells. Journal of Clinical Lipidology 2017 11, 694-703DOI: (10.1016/j.jacl.2017.03.007) Copyright © 2017 National Lipid Association Terms and Conditions

Figure 4 Loss of circadian release of diabetic CD34+ CACs. Peripheral blood was collected every 2 hours for a total of 24 hours from (A) control (n = 4) and (B) diabetic (n = 8) subjects and was analyzed for the number of CD34+ CACs by flow cytometry. (A) In control subjects, there is a clear peak of circulating CD34+ CACs that occurred in the middle of the night. (B) Rhythmic CD34+ CACs release pattern was blunted in type II diabetic patients. The dash line is the model fitted curve for individual patients and bold curve is the fitted curve for population. CACs, circulating angiogenic cells. Journal of Clinical Lipidology 2017 11, 694-703DOI: (10.1016/j.jacl.2017.03.007) Copyright © 2017 National Lipid Association Terms and Conditions