Gb3 loading and alpha-Galactosidase A gene silencing.

<p><b>A.</b> Incubation with Gb3 increased intracellular concentration of Gb3, as measured by ELISA, by 2-folds in HMaVECs and HMiVECs (n = 4, p = 0.03 and p = 0.04, respectively, versus untreated control, <b>B.</b> Maximal a-Gal A protein silencing of 70% occurred 48 hrs after transfection of HMaVECs with siRNA 3. Transfection of HMaVECs with Non-targeting (NT)-RNA did not alter endothelial a-Gal A expression. Values are representative of at least 4 different experiments; all blots are normalized to GAPDH expression. <b>C.</b> Intracellular Gb3 concentrations 2 days following siRNA transfection remained unchanged compared to untreated cells.</p

Gb3 enhances VCAM-1 expression in TNF-α stimulated HMiVECs.

<p><b>A.</b> TNF-α enhances VCAM-1 expression in HMiVECs. Values are indicated as percent of unstimulated control. *p<0.05 vs unstimulated control; Values are representative of at least 4 different experiments; all blots are normalized to GAPDH expression. <b>B.</b> Gb3 enhances VCAM-1 expression in TNF-α stimulated HMaVECs and under basal conditions. Values are indicated as percent of unstimulated control. *p<0.05 vs unstimulated control; **p<0.05 vs TNF-α alone. <b>C./D.</b> GLA gene silencing did not affect expression of VCAM-1.</p

Gb3 impairs eNOS expression in HMiVECs.

<p><b>A.</b> Gb3 inhibits total eNOS expression in TNF-α stimulated HMiVECs and under basal conditions. Values are indicated as percent of unstimulated control. *p<0.05 vs unstimulated control; **p<0.05 vs TNF-α alone. <b>B.</b> Gb3 inhibits Ser₁₁₇₇ pho eNOS expression in TNF-α stimulated HMiVECs and under basal conditions. Values are indicated as percent of unstimulated control. *p<0.05 vs unstimulated control; **p<0.05 vs TNF-α alone. Values are representative of at least 4 different experiments; all blots are normalized to GAPDH expression. <b>C./D.</b> In all cases expression remained unchanged following GLA gene silencing.</p

Gb3 enhances COX-2 expression in HMiVECs.

<p><b>A.</b> Gb3 enhances COX-2 expression in HMiVECs under basal conditions. TNF-α enhances COX-2 expression in HMiVECs. Values are indicated as percent of unstimulated control. *p<0.05 vs unstimulated control. <b>B.</b> TNF-α enhanced COX-1 expression in both HMiVECs and HMaVECs by 1.5-fold and 1.4-fold, respectively. Values are representative of at least 4 different experiments; all blots are normalized to GAPDH expression. <b>C.</b> GLA gene silencing had no effect on expression of COX-2.</p

Gb3 enhances iNOS expression in HMiVECs.

<p><b>A.</b> Gb3 enhances iNOS expression in HMiVECs under basal conditions. TNF-α enhances iNOS expression in HMiVECs. Values are indicated as percent of unstimulated control. *p<0.05 vs unstimulated control. <b>B.</b> TNF-α enhances iNOS expression in HMaVECs. Values are indicated as percent of unstimulated control. *p<0.05 vs unstimulated control; Values are representative of at least 4 different experiments; all blots are normalized to GAPDH expression. <b>C./D.</b> GLA gene silencing had no effect on expression of iNOS.</p