Sun, Ruohan

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.

Background and Purpose: Molecular mechanisms underlying increased risk of Alzheimer’s disease in hypertensive patients are poorly defined. Conflicting results have been reported regarding the effects of angiotensin II (Ang II) on expression of amyloid precursor protein (APP) and enzymes responsible for proteolytic cleavage of APP in the brain. However, no previous studies have directly assessed the effects of Ang II on metabolisms of APP in human endothelial cells. Current study was designed to determine the effects of Ang II on the expression and processing of APP in cultured human brain microvascular endothelial cells (hBMECs). Methods: Human BMECs were treated with increasing concentrations of Ang II (0.1nM to 100nM; incubation time = 24h). The protein expression of APP, α-secretase [a disintegrin and metalloprotease 10, (ADAM10), ADAM9, and ADAM17], β-secretase (β-site APP-cleaving enzyme, BACE1), NADPH oxidase subunits and endothelial nitric oxide synthase (eNOS) were measured by western blot. Production and release of amyloid β-peptides (Aβ) from endothelium were detected by ELISA. Results: In hBMECs, Ang II had no effect on the expression of APP (n=7-9, P>0.05) and α-secretase (ADAM10, n=7-9, P>0.05; ADAM9, n=4-7, P>0.05; ADAM17, n=4-7, P>0.05). Treatment with Ang II at 1nM concentration induced significant increase in BACE1 expression (n=13, P<0.05), however, we did not observe significant changes in production of Aβ40 (n=11, P>0.05) or Aβ42 (n=11, undetectable). The higher concentration of Ang II (100nM) increased expression of NOX4 (n=6, P<0.05). In addition, Ang II (1nM and 10nM) significantly up-regulated eNOS protein levels (n=8-10, P<0.05). Conclusion: Our findings suggest that in hBMECs: a) Ang II does not affect expression of APP and α-secretase-mediated cleavage of APP, and b) Ang II increases expression of BACE1 but does not elevate production and release of Aβ peptides. We also confirm previously reported ability of Ang II to increase expression of NOX4 and eNOS. We speculate that under pathological conditions, increased expression of BACE1 induced by Ang II may predispose cerebrovascular endothelium to elevated amyloidogenic processing of APP.

The mechanisms underlying proangiogenic function of brain-derived neurotrophic factor (BDNF) are not fully understood. The current study was designed to explore the microRNA (miRNA) profile in human early endothelial progenitor cells (EPCs, also referred to as CFU-Hill cells) treated with BDNF. Treatment of early EPCs with BDNF for 7 d significantly increased the colony formation of outgrowth endothelial cells. BDNF suppressed the expression of miR-4716-5p, miR-3928, miR-433, miR-1294, miR-1539, and miR-19b-1*. In contrast, BDNF significantly increased the levels of miR-432*, miR-4499, miR-3911, miR-1183, miR-4669, miR-636, miR-4717-3p, miR-4298, miR485-5p, and miR-181c. Since miR-433 has been reported to augment hematopoietic cells proliferation and differentiation, we examined the role of miR-433 in regenerative effects of BDNF. BDNF stimulated the protein expression of guanylate-binding protein 2 via the suppression of miR-433. However, the knockdown of miR-433 was not sufficient to significantly increase the number of outgrowth endothelial cell colonies, suggesting that modulation of miR-433 alone does not stimulate regenerative capacity of EPCs. In aggregate, our results also suggest that the effect of BDNF on regenerative function of EPCs may depend on complex changes in the expression of microRNAs.

The mechanisms underlying dysfunction of cerebral microvasculature induced by type 1 diabetes (T1D) are not fully understood. We hypothesized that in cerebral microvascular endothelium, α-processing of amyloid precursor protein (APP) is impaired by T1D. In cerebral microvessels derived from streptozotocin (STZ)-induced T1D mice protein levels of APP and its α-processing enzyme, a disintegrin and metalloprotease 10 (ADAM10) were significantly decreased, along with down-regulation of adenylate cyclase 3 (AC3) and enhanced production of thromboxane A 2 (TXA 2 ). In vitro studies in human brain microvascular endothelial cells (BMECs) revealed that knockdown of AC3 significantly suppressed ADAM10 protein levels, and that activation of TXA 2 receptor decreased APP expression. Furthermore, levels of soluble APPα (sAPPα, a product of α-processing of APP) were significantly reduced in hippocampus of T1D mice. In contrast, amyloidogenic processing of APP was not affected by T1D in both cerebral microvessels and hippocampus. Most notably, studies in endothelial specific APP knockout mice established that genetic inactivation of APP in endothelium was sufficient to significantly reduce sAPPα levels in the hippocampus. In aggregate, our findings suggest that T1D impairs non-amyloidogenic processing of APP in cerebral microvessels. This may exert detrimental effect on local concentration of neuroprotective molecule, sAPPα, in the hippocampus.