High glucose promotes A20 O-glycosylation, ubiquitination and proteasomal degradation in EC and SMC.

<p>High glucose increases protein O-GlcNAcylation, including that of A20 and possibly other E3 Ubiquitin ligases. This leads to increased A20 ubiquitination either through auto-ubiquitination or increased activity of other O-GlcNAcylated E3 ubiquitin ligases. This targets A20 for degradation in the proteasome. Blockade of O-GlcNAcylation using DON, upstream of A20 Ubiquitination, or inhibition of proteasome activity, using MG132, downstream of A20 ubiquitination would inhibit its proteasomal degradation, restoring its expected protein levels.</p

LPS-mediated upregulation of A20 protein expression is blunted in aortae of diabetic, as compared to non-diabetic mice.

<p>(A) WB of A20 in abdominal aortae of diabetic and non-diabetic atherosclerosis-prone C57BL/6 and atherosclerosis-resistant FVB/N mice, 8 h after LPS treatment. GAPDH and βactin were used to correct for loading and quantify relative A20 expression by densitometry, as reported below the WB. Data shown are representative of 3 (non-diabetic) and 4 (diabetic) mice per time-point and illustrate the loss of LPS-induced A20 protein in diabetic mice, regardless of strain. The cuts between samples reflect the fact that these samples, while on the same gel and same experiment, were not contiguous. (B) A20 mRNA levels analyzed by real-time PCR 3 to 8 h after LPS injection in mouse abdominal aortae (n = 5 non-diabetic and 7 diabetic mice in C57BL/6 and 3 non-diabetic and 4 diabetic mice in FVB/N). Data shown demonstrates that LPS increases A20 mRNA levels in aortae of diabetic and non-diabetic C57BL/6 and FVB/N, albeit at a greater levels in diabetic mice. Expression of 18S ribosomal RNA was used to normalize expression of A20 mRNA, and the results were presented as mean±SEM of mRNA. Each sample was measured in duplicate.</p

Restoring A20 levels reverts glucose-mediated upregulation of RAGE and phosphorylation of PKCβII.

<p>(A) WB analysis for RAGE and A20 expression in SMC cultured in 5 or 30 mM D-Glu for 24 h and treated with TNF in the presence or absence of 20 mM of Azaserine (prior to TNF) or 10 mM of MG132 (following TNF). Corrected RAGE fold-inductions are listed below the WB. The RAGE protein is detected as a doublet as a result of pre and post-N-glycosylated form of the protein. Both bands were used for densitometry evaluation. (B) WB analysis of phospho-PKCβII (pPKCβII) and total (c) PKCβII in NT SMC, and in SMC transduced with rAd.A20 or rAd.βgal, and treated with PMA or challenged with 30 mM D-Glu for 1 h. Data shown in A and B are representative of 3 independent experiments. NT = non-transduced cells. GAPDH was used as loading control to quantify the relative expression of RAGE and pPKCβII by densitometry.</p

Increasing glucose (D-Glu) concentrations decreases TNF-mediated A20 protein up-regulation without affecting its transcriptional activation in SMC.

<p>(A) Analysis of A20 expression by WB. SMC were cultured in 5, or 30 mM D-Glu, or the osmotic control (25 mM L-Glu +5 mM D-Glu), and treated with TNF for 6 h. βactin was checked as a loading control and used to quantify relative A20 expression by densitometry, as reported beneath the WB. Densitometry of the bands of interest and was determined as the mean intensity of the areas delineated by Image J, then corrected by the main intensity of the corresponding housekeeping gene band. Fold induction was determined using the non-treated 5 mM D-glucose condition sample as one (1). Data are representative of 3 independent experiments. The cuts between samples reflect the fact that these samples, while on the same gel and same experiment, were not contiguous. (B) Analysis of A20 mRNA levels by real-time PCR. SMC were cultured in 5 or 30 mM D-Glu or mannitol (25 mM Mannitol +5 mM D-GLu), as an osmotic control, and treated with TNF for 1 or 3 h. 18S ribosomal RNA was used to normalize the data. Natural log transformed data (ln) are presented as mean±SEM of 3 independent experiments performed in duplicate. No significant differences (P>0.05) were noted between all groups and at all time-points.</p

O-GlcNAcylation and ubiquitination of A20 modulate its expression.

<p>(A) WB analysis of total A20 and co-immunoblotted, overlapping GlcNAc-A20 (RL-2) in SMC cultured in 5, 15 and 30 mM of D-glucose (D-Glu), and treated or not with TNF for 6 h. β-actin was used as a control for loading. (B) WB analysis of WGA captured proteins from SMC cultured in 5 and 30 mM D-Glu demonstrate the presence of glycosylated (GlcNAcA20), and co-immunoblotted, overlapping, ubiquitinated A20 (Ub-A20). (C) WB analysis of cell lysates immunoprecipitated with the A20 antibody from SMC cultured in 5 and 30 mM of D-Glu and treated or not with TNF for 6 h, and analyzed WB for total A20 and GlcNAc-A20 using the RL2 antibody demonstrate increased GlcNAc-A20 in high glucose medium. (D) WB analysis of cell lysates immunoprecipitated with the A20 antibody from SMC cultured in 5 and 30 mM D-Glu and treated or not with TNF for 6 h, and analyzed by WB for total and Ub-A20 demonstrate increased Ub-A20 in high glucose medium. (E) WB analysis of total and overlapping GlcNAc-A20 (RL-2) in SMC cultured in 30 mM D-Glu and treated with DON (prior to TNF) or MG132 (after TNF). (F) WB analysis of total and phospho-A20 in SMC cultured in 5, 15 and 30 mM D-Glu and treated with TNF for 6 h demonstrated that relative phosphorylation levels of A20 (pA20) were not decreased by high glucose, despite decreased TNF-mediated upregulation of A20 protein in cells cultured in high glucose. GAPDH or βactin was checked as a loading control to quantify A20 expression by densitometry. Corrected A20 fold-inductions are listed below the WB. RL2/A20 and Ubiquitin/A20 ratios were also calculated by densitometry. Data shown in A, C, D, and E are representative of 3 independent experiments. Data shown in B and F are representative of 2 independent experiments.</p

Intravenous administration of rAd.A20 to diabetic ApoE-null mice fails to protect them from accelerated atherosclerosis.

<p>Evaluation of I/M ratio on H&E stained aortic arch sections from 20-week old mice (8 weeks following intravenous administration of 2.5×10⁹ pfu/mice of rAd.A20 or saline) demonstrated comparable intimal lesions in saline and rAd.A20-treated mice (n = 6 mice/group). Images are shown at the level of the first coronary artery at 100× magnification. The asterisk indicates the level of the first coronary artery branch. Arrows define the intima (I). Immunohistochemistry analysis of RAGE and phospho-PKCβII (pPKCβII) demonstrate equally intense staining in the neointima of saline and rAd.A20-treated mice. Image amplification 400×.</p

Expression of A20 in the ascending aorta and aortic arch of diabetic ApoE-null mice prevents the development of atherosclerotic lesions by inhibiting PKCβII phosphorylation and blunting the induction of RAGE.

<p>(A) Transgene expression was confirmed by X-gal staining in rAd.βgal-transduced vessels 5 days following transgene delivery (n = 3 mice/group) and demonstrate the expression of the transgene in medial SMC (M) as well as the adventitia at the level of the aortic root, albeit not in all cells. Image amplification 100× and 400×. (B) A20 expression was verified by real time RT-PCR in two rAd.A20-transduced vessels, using human A20 specific primers that do not recognize mouse A20. Our data indicate significant expression of human A20 in aortic roots of rAd.A20 but not saline treated mice. Results are shown as average± SE. (C) H&E stained aortic root sections at the level of the first coronary from 20 week-old diabetic ApoE-null mice treated with saline, rAd.A20 or rAd.βgal. Images are shown at 100× and 400× as indicated by the scale bar. The asterisk indicates the level of the first coronary branch, Arrows define the intima (I) and the media (M). ApoE-competent, non-diabetic C57BL/6 and non-diabetic ApoE-null mice were used as controls. Blood glucose and cholesterol levels (cholest) are listed below the sections. *P<0.05 compared to saline, ** P<0.01 compared to rAd. βGal. Data shown are representative of 4 to 6 mice per group. I/M ratios were calculated after analysis of 10 serial sections per vessel. (D) phospho-(p)PKCβII (5 days) and RAGE (14 days) immunostaining in aortic arches 5 and 14 days after transgene delivery. Data shown in C are representative of all sections analyzed (n = 3 mice per group, 2–3 sections analyzed per vessel). Image amplification 200×.</p

Increasing glucose (D-Glu) concentrations decreases TNF-mediated A20 protein up-regulation without affecting its transcriptional activation in EC.

<p>(A) Analysis of A20 expression by WB. EC cultured in medium containing 5, 15 or 30 mM D-glucose (D-Glu) or L-glucose (L-Glu), as an osmotic control, were stimulated with TNF for 6 h˜βactin was checked as loading control and to quantify relative A20 expression by densitometry, as reported below the WB. Densitometry of the bands of interest and was determined as the mean intensity of the areas delineated by Image J, then corrected by the main intensity of the corresponding house keeping gene band. Fold induction was determined using the non-treated 5 mM D-glucose condition sample as one (1). A20 protein migrates as a doublet in EC and hence both bands were scanned. Data are representative of 3 independent experiments. (B) Analysis of A20 mRNA levels by real-time PCR. EC cultured in medium containing 5, 15 or 30 mM D-Glu or L-Glu as an osmotic control, were stimulated with TNF for 1 and 3 h. Expression of 18S ribosomal RNA was used to normalize the expression of A20 mRNA. Natural log transformed data (ln) are presented as mean±SEM of 3 independent experiments performed in duplicate. No significant differences (P>0.05) were observed between all groups and at all time-points.</p