Mechanisms of Diabetes-Induced Endothelial Cell Senescence: Role of Arginase 1
<p>Diabetes induces increases in arginase 1 (A1) expression. (<b>A</b>) Western blot analysis and quantification (<b>B</b>) showing increased A1 expression in wild type (WT) diabetic retinas. ** <span class="html-italic">p</span> < 0.01 vs. WT Ctrl. <span class="html-italic">n</span> = 3–4; (<b>C</b>) Western blot analysis and quantification (<b>D</b>) show similar levels of A2 expression in WT diabetic mice and age matched controls. <span class="html-italic">p</span> = 0.4, <span class="html-italic">n</span> = 4.</p> "> Figure 2
<p>Diabetes induces senescence in retinal tissue. Senescence associated β-galactosidase (SA β-Gal) activity images of frozen retinal sections showing positive signal in cells of the large vessels (white arrows) as well as in the surrounding tissues (red arrows) of the central retina. Scale bar = 20 µM.</p> "> Figure 3
<p>Arginase inhibition prevents diabetes-induced alterations in retinal senescence markers. qRT-PCR showing increased mRNA levels of p16 (<b>A</b>); p21 (<b>B</b>) p53 (<b>C</b>); and, Igfbp3 (<b>D</b>) in the diabetic retinas compared to age matched controls. 2(S)-Amino-6-boronohexanoic acid (ABH) treatment significantly blocked the diabetes-induced senescence. * <span class="html-italic">p</span> < 0.05 vs. WT Ctrl, # <span class="html-italic">p</span> < 0.05 vs. WT DB. <span class="html-italic">n</span> = 4–9.</p> "> Figure 4
<p>Arginase inhibition reduces p16 expression in isolated vessels. Immuno-labeling of isolated retinal vessels showing expression of the senescence protein p16 (green) in vascular endothelial cells which was co-localized with the endothelial marker (isolectin B4, red). Diabetic vessels showed increased p16 expression compared to controls. Arginase inhibition with ABH significantly reduced p16 expression. <span class="html-italic">n</span> = 3–4. Scale bar = 50 µM.</p> "> Figure 5
<p>Diabetes induced- increases in phospho p38 is prevented by inhibiting arginase. (<b>A</b>) Western blot analysis and quantification (<b>B</b>) showing increased phosphorylation of p38 in WT diabetic retinal extracts compared to age matched controls. ABH treatment significantly blocked this increase. * <span class="html-italic">p</span> < 0.05 vs. WT Ctrl. # <span class="html-italic">p</span> < 0.001 vs. WT DB. <span class="html-italic">n</span> = 6–8.</p> "> Figure 6
<p>A1 deletion prevents diabetes-induced senescence in retinal vessels. SA β-Gal activity images of isolated retinal vessels showing increased activity (arrows) in vessels isolated from WT diabetic mice. The vessels isolated from A1+/− diabetic retinas are negative for SA β-Gal staining. The non-diabetic control retinas from both WT and A1+/− mice are also negative for SA β-Gal staining. <span class="html-italic">n</span> = 3–5. Scale bar = 50 µM.</p> "> Figure 7
<p>High glucose-induces endothelial cell senescence. (<b>A</b>) Senescence associated β-Gal activity (black arrows) and quantification (<b>B</b>) showing a significant increase in SA β-Gal positive cells with high glucose (HG, 25 mM) treatment when compared to normal glucose (NG, 5 mM). ABH treatment prevented this increase. * <span class="html-italic">p</span> < 0.05 vs. NG. # <span class="html-italic">p</span> < 0.05 vs. HG. <span class="html-italic">n</span> = 3. Scale bar = 50 µM; (<b>C</b>) Western blot analysis with quantification (<b>E</b>) showing increased p16<sup>INK4A</sup> expression in HG treated bovine retinal endothelial cells (BRECs) as compared to NG. Arginase inhibition with ABH blocked this increase. **** <span class="html-italic">p</span> < 0.0001 vs. NG. # <span class="html-italic">p</span> < 0.0001 vs. HG. <span class="html-italic">n</span> = 3. Experiments were repeated at least twice in independent batches of cells. Data are presented as a fold change of the NG; and, (<b>D</b>) Western blot analysis with quantification (<b>F</b>) showing increased phosphorylation of p38 in HG treated BRECs as compared to NG. Arginase inhibition with ABH significantly blocked this increase. # <span class="html-italic">p</span> < 0.05 vs. HG. <span class="html-italic">n</span> = 3. Experiments were repeated at least twice in independent batches of cells. Data are presented as a fold change of the NG.</p> "> Figure 8
<p>Arginase 1 (A1) overexpression induces endothelial cell senescence. (<b>A</b>) Western blot analysis with quantification (<b>B</b>) showing levels of A1 protein or (<b>C</b>) phosphorylation of the stress marker p38 in bovine retinal endothelial cells (BRECs) transduced with 20 MOI of wild type A1 (A1), inactive mutant A1 (Δ A1), or red fluorescence protein (RFP) adenovirus vectors. The inactive mutant A1 was generated by mutation of aspartic acid 128 to glycine. (<b>B</b>) ** <span class="html-italic">p</span> < 0.01 vs. RFP. *** <span class="html-italic">p</span> < 0.001 vs. RFP. <span class="html-italic">n</span> = 6–8. (<b>C</b>) n.s., non-significant, ** <span class="html-italic">p</span> < 0.01 vs. RFP & Δ A1. <span class="html-italic">n</span> = 3–8. Experiments were repeated at least twice in independent batches of cells. Data are presented as a fold change of RFP; (<b>E</b>) Senescence associated β-galactosidase (SA β-Gal) activity with quantification (<b>D</b>) showing a significant increase in SA β-Gal positive cells with A1 overexpression as compared to RFP or the inactive mutant A1 (Δ A1). * <span class="html-italic">p</span> < 0.05 vs. RFP & Δ A1. <span class="html-italic">n</span> = 5–10. Experiments were repeated at least twice in independent batches of cells. Data are presented as a fold change of RFP. Scale bar = 50 µM.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Diabetes-Induced Increases in Arginase 1 and Premature Cellular Senescence
2.2. Suppression of Diabetes-Induced Increases in Senescence Markers by Inhibition of Arginase
2.3. Induction of Endothelial Cell Senescence by High Glucose or Arginase 1 Overexpression and Blockade by Arginase Inhibition
3. Discussion
4. Materials and Methods
4.1. Animals and Diabetes Induction
4.2. Retinal Vessels Isolation
4.3. Cell Culture Studies
4.4. PCR Analyses
4.5. Western Blot
4.6. SA β-Galactosidase Activity
4.7. Immunofluorescence Analysis of Isolated Retinal Vessels
4.8. Statistical Analysis
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
A1 | Arginase1 |
A1+/− | A1 heterozygous knockout |
A2 | Arginase 2 |
ABH | [2(S)-Amino-6-boronohexanoic acid], arginase inhibitor |
AGE | Advanced glycation end products |
BRECs | Bovine retinal endothelial cells |
BSA | Bovine serum albumin |
DAPI | 4′,6′-diamino-2-phenylindole |
ECs | Endothelial cells |
FBS | Fetal bovine serum |
HG | High glucose |
HPRT | Hypoxanthine phosphoribosyl transferase |
HUVECs | Human umbilical vein endothelial cells |
Igfbp3 | Insulin-like growth factor binding protein-3 |
MOI | Multiple of infections |
NG | Normal glucose |
NO | Nitric oxide |
NOS | Nitric oxide synthase |
NOX2 | NADPH oxidase 2 |
OCT | Optimal cutting temperature |
P16INK4a | cyclin-dependent kinase inhibitor 2A |
P21 | cyclin-dependent kinase inhibitor 1 |
P53 | Tumor suppressor p53 |
PBS | Phosphate-buffered Saline |
RFP | Red fluorescence protein |
SA β-Gal | Senescence associated β-galactosidase |
STZ | Streptozotocin |
WT | Wild type |
WT Ctrl | Wild type control |
WT DB | Wild type diabetic |
Δ A1 | Inactive mutant arginase 1 |
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Shosha, E.; Xu, Z.; Narayanan, S.P.; Lemtalsi, T.; Fouda, A.Y.; Rojas, M.; Xing, J.; Fulton, D.; Caldwell, R.W.; Caldwell, R.B. Mechanisms of Diabetes-Induced Endothelial Cell Senescence: Role of Arginase 1. Int. J. Mol. Sci. 2018, 19, 1215. https://doi.org/10.3390/ijms19041215
Shosha E, Xu Z, Narayanan SP, Lemtalsi T, Fouda AY, Rojas M, Xing J, Fulton D, Caldwell RW, Caldwell RB. Mechanisms of Diabetes-Induced Endothelial Cell Senescence: Role of Arginase 1. International Journal of Molecular Sciences. 2018; 19(4):1215. https://doi.org/10.3390/ijms19041215
Chicago/Turabian StyleShosha, Esraa, Zhimin Xu, S. Priya Narayanan, Tahira Lemtalsi, Abdelrahman Y. Fouda, Modesto Rojas, Ji Xing, David Fulton, R. William Caldwell, and Ruth B. Caldwell. 2018. "Mechanisms of Diabetes-Induced Endothelial Cell Senescence: Role of Arginase 1" International Journal of Molecular Sciences 19, no. 4: 1215. https://doi.org/10.3390/ijms19041215