Inactivation of BACE1 increases expression of endothelial nitric oxide synthase in cerebrovascular endothelium

Abstract

Cerebrovascular effects of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) inactivation have not been systematically studied. In the present study we employed cultured human brain microvascular endothelial cells (BMECs), BACE1-knockout (BACE1^-/-) mice and conditional (tamoxifen-induced) endothelium-specific BACE1-knockout (eBACE1^-/-) mice to determine effect of BACE1 inhibition on expression and function of endothelial nitric oxide synthase (eNOS). Deletion of BACE1 caused upregulation of eNOS and glypican-1 (GPC1) in human BMECs treated with BACE1-siRNA, and cerebral microvessels of male BACE1^-/- mice and male eBACE1^-/- mice. In addition, BACE1siRNA treatment increased NO production in human BMECs. These effects appeared to be independent of amyloid β-peptide production. Furthermore, adenoviral-mediated overexpression of BACE1 in human BMECs down-regulated GPC1 and eNOS. Treatment of human BMECs with GPC1siRNA suppressed mRNA and protein levels of eNOS. In basilar arteries of male eBACE1^-/- mice, endothelium-dependent relaxations to acetylcholine and endothelium-independent relaxations to NO donor, DEA-NONOate, were not affected, consistent with unchanged expression of eNOS and phosphorylation of eNOS at Ser1177 in large cerebral arteries. In aggregate, our findings suggest that under physiological conditions, inactivation of endothelial BACE1 increases expression of eNOS in cerebral microvessels but not in large brain arteries. This effect appears to be mediated by increased GPC1 expression.

Keywords: Alzheimer’s disease; BACE1; eNOS; endothelial cells; glypican-1.

Figure 1.

BACE1 regulated eNOS expression and NO production in human BMECs. A-F: Cells were treated with BACE1siRNA or CtsiRNA (30 nM) for 48 hours (a and c–f) or 40 hours (b), Western blot or real-time PCR were performed, respectively. a, BACE1 protein was knocked down using BACE1siRNA, n = 8 *P < 0.05. b, levels of eNOS mRNA were increased in cells treated with BACE1siRNA, n = 4, *P < 0.05. c–f, protein expressions of eNOS, p-eNOS(S1177)/eNOS, and p-eNOS(T495)/eNOS) in human BMECs treated BACE1siRNA or CtsiRNA, n = 15-24, *P < 0.05. g and h: Human BMECs were transduced with Ad-BACE1 or Ad-Null (2.5 MOI, 2 days). g, n = 9, H, n = 13; *P < 0.05. i: Human BMECs were treated with BACE inhibitor IV for 24 hours (n = 5, *P < 0.05). J: After endothelial cells were treated with 30 nM BACE1siRNA for 2 days, cells were incubated with EGM2 without serum for 24 hours. The supernatants were collected for NO₂+NO₃ assay (n = 8, *P < 0.05).

Figure 1.

BACE1 regulated eNOS expression and NO production in human BMECs. A-F: Cells were treated with BACE1siRNA or CtsiRNA (30 nM) for 48 hours (a and c–f) or 40 hours (b), Western blot or real-time PCR were performed, respectively. a, BACE1 protein was knocked down using BACE1siRNA, n = 8 *P < 0.05. b, levels of eNOS mRNA were increased in cells treated with BACE1siRNA, n = 4, *P < 0.05. c–f, protein expressions of eNOS, p-eNOS(S1177)/eNOS, and p-eNOS(T495)/eNOS) in human BMECs treated BACE1siRNA or CtsiRNA, n = 15-24, *P < 0.05. g and h: Human BMECs were transduced with Ad-BACE1 or Ad-Null (2.5 MOI, 2 days). g, n = 9, H, n = 13; *P < 0.05. i: Human BMECs were treated with BACE inhibitor IV for 24 hours (n = 5, *P < 0.05). J: After endothelial cells were treated with 30 nM BACE1siRNA for 2 days, cells were incubated with EGM2 without serum for 24 hours. The supernatants were collected for NO₂+NO₃ assay (n = 8, *P < 0.05).

Figure 2.

GPC1 mediated effect of BACE1 on eNOS expression in human BMECs. a and b: Cells were treated with 30 nM BACE1siRNA or CtsiRNA for 48 hours (a) or 40 hours (b), the protein or mRNA levels of GPC1 were measured, respectively. (a) n = 12; (b) n = 4; *P < 0.05. PC-12 cell lysate was used as a GPC1 positive control. (c) Cells were transduced with Ad-BACE1 or Ad-Null (2.5 MOI, 2 days). n = 8, *P < 0.05. (d–f) Cells were treated with 30 nM GPC1siRNA or CtsiRNA for 2 days. mRNA expressions of GPC1 (d) and eNOS (e), as well as eNOS protein levels (f) were measured. D, n = 6; E, n = 7, F, n = 4; *P < 0.05.

Figure 3.

Expressions of eNOS and GPC1 in brain microvessels of BACE1^−/− mice were increased. a–d: Cerebral microvessels were isolated from BACE1^−/− mice and wild type littermates and subjected to Western blot analysis. n = 8, *P < 0.05.

Figure 4.

Expressions of eNOS and GPC1 in brain microvessels of eBACE1^−/− mice were augmented. (a) Representative genotyping analysis of eBACE1^−/− mouse (BACE1^flox/flox;Cdh5-Cre⁺) and WT littermate (BACE1^flox/flox;Cdh5-Cre⁻). (b) PCR analysis of BACE1 in mouse aorta and endothelium isolated from mouse aorta. Notice that BACE1 was deleted from aortic endothelium in eBACE1^−/− mice. (c–f): Protein levels of BACE1, eNOS, and GPC1 in brain microvessels of eBACE1^−/− mice and WT mice. n = 7–10, *P < 0.05.

Figure 5.

Concentration-dependent contractions in basilar arteries to U46619 (a, n = 6, *P < 0.05) and ET-1 (b, n = 5–6, *P < 0.05). Endothelium-dependent relaxations in basilar arteries to acetylcholine (c, n = 6, P > 0.05) and DEA-NONOate (d, n = 6, P > 0.05). e–g: Protein expressions of eNOS and p-eNOS(s1177) in large cerebral arteries were not significantly changed. Quantification of expressions of eNOS (f, n = 11, P > 0.05) and ratio of p-eNOS(1177)/eNOS (g, n = 7, P > 0.05).