He, Tongrong

Objectives- Oxidative stress and endothelial dysfunction precedes cerebral β-amyloid deposits in human Alzheimer’s disease as well as in transgenic mouse models expressing mutations in amyloid precursor protein (APP). In the present study, we hypothesized that uncoupling of endothelial nitric oxide synthase (eNOS) caused by loss of its essential co-factor, tetrahydrobiopterin (BH 4 ), in cerebral arteries and microvessels contributes, in part, to oxidative stress and cerebral vascular dysfunction in a APP transgenic mouse (Tg2576) that express the Swedish double mutation of human APP. In addition, we examined whether treatment with Peroxisome Proliferator Activated Receptor-δ (PPARδ) activator - GW501516 restores bioavailability of BH 4 and reverses oxidative stress in APP Tg2576 mice. Methods- APP Tg2576 mice (4-5 months old) were treated with GW501516 , a selective PPARδ activator (2 mg/kg/day, po, 14 days). Following treatment, cerebral arteries and microvessels were obtained. Biopterin levels, enzymatic activity of GTP cyclohydrolase I (GTPCH I) and superoxide production were measured by HPLC and protein expression was studied by Western blotting. Results- Cerebral arteries and microvessels demonstrated increased expression of eNOS, while the bioavailability of its essential co-factor BH 4 was significantly reduced, suggestive of eNOS uncoupling in APP Tg2576 mice. Furthermore, expressions of catalase and manganese superoxide dismutase (MnSOD) were decreased, while superoxide production was increased (P<0.01, n=6) in cerebral microvessels of APP Tg2576 mice. Treatment with GW501516 restored the BH 4 /BH 2 ratio in cerebral arteries and microvessels of APP Tg2576 mice (P<0.05, n-7-8), while the enzymatic activity of GTPCH-I remained unchanged (P>0.05, n=6). PPARδ activation also prevented the attenuation in expressions of MnSOD and catalase, and inhibited the increased superoxide production (P<0.05, n=6) in cerebral arteries and microvessels of APP Tg2576 mice. Interestingly, PPARδ activation significantly inhibited the over-expression of APP in cerebral microvessels obtained from APP Tg2576 mice (P<0.001, n=4). Conclusion- Our results suggest that endothelial dysfunction in APP Tg2576 mice may be caused, in part, by reduced bioavailability of BH 4 and uncoupling of eNOS. Treatment of APP Tg2576 mice with PPARδ agonist GW501516 exerted cerebral vascular protection by multiple mechanisms: (a) by inhibiting eNOS uncoupling via increased expressions of MnSOD and catalase, as well as, (b) by attenuating the increased expression of APP in the cerebral microvessels of APP Tg2576 mice.

Previous studies have established that cerebral blood vessels possess high capacity to synthesize prostacyclin (PGI 2 ). Substantial evidence indicates that amyloid precursor protein (APP) and soluble APPα (sAPPα) have neurotrophic and nueroprotective properties. We hypothesized that activation of IP-cAMP pathway stimulates non-amyloidogenic processing of APP in cerebral endothelial cells. Western blotting was used to detect protein expression; real time RT-PCR was employed to measure mRNA levels of APP, and a disintegrin and metalloprotease 10 (ADAM10, an α processing enzyme); sAPPα secretion was detected in condition medium derived from culturing human brain microvascular endothelial cells (BMVECs), by ELISA. After human BMVECs were treated with PGI 2 analog iloprost (0.01, 0.05, 0.1, or 0.5 μM) for 3 days, protein levels of ADAM10 (n=7-9, P<0.05) and APP (n=6, P<0.05) were significantly increased in a concentration dependent manner, while β amyloidogenic processing enzyme BACE1, and other α processing enzymes such as ADAM9 and ADAM17 were not changed. mRNA levels of APP (n=7, P<0.05) and ADAM10 (n=4, P<0.05), as well as sAPPα production in human BMVECs (n=6, P<0.05) were also significantly augmented in response to iloprost treatment. Moreover, stimulation of human BMVECs with forskolin (40μM) significantly enhanced APP and ADAM10 protein expression. Most notably, iloprost significantly augmented protein expression of peroxisome proliferation-activated receptor-δ (PPARδ) and SIRT1. We further explored the role of PPARδ in APP processing. ADAM10 expression (n=6, P<0.05), but not APP and BACE1, was significantly increased in human BMVECs treated with PPARδ ligand GW501516 (30 or 100 nM). GW501516 (100 nM) also significantly increase sAPPα production (n=9-10, P<0.05). siRNA against PPARδ significantly suppressed basal levels of ADAM10, and blocked iloprost-induced ADAM10 expression (n=4, P<0.05). These findings suggest that activation of IP-cAMP pathway enhances non-amyloidogenic processing of APP and consequently increases production of neurotrophic molecule sAPPα in human BMVECs. Activation of PPARδ in part mediates iloprost-induced α processing of APP.

Under physiological conditions, β-site amyloid precursor protein (APP)-cleaving enzyme 2 (BACE2) cleaves APP within Aβ sequence thereby functioning like a α-secretase. However, BACE2 could also function as a conditional β-secretase during aging, contributing to Alzheimer’s disease pathogenesis. To date the physiological functions of BACE2 in endothelium are largely unknown. The present study is therefore designed to investigate the role of BACE2 in APP metabolism in human BMECs. Cultured human BMECs (passage 5-6 or passage 22) were treated with BACE2siRNA (30 nM, for 3 days), levels of soluble APPα (sAPPα, a neurotrophic product of non-amyloidogenic processing of APP) and Aβ40 in the supernatant were measured. In human BMECs (passage 5-6), genetic inactivation of BACE2 significantly decreased production of sAPPα (n=12, P<0.05), but had no effect on production of Aβ40 (n=9, P>0.05). BACE2siRNA treatment significantly suppressed APP protein expression (n=7, P<0.05), but augmented protein levels of BACE1 (n=7, P<0.05). Genetic inactivation of BACE2 did not change protein levels of mature ADAM10 (n=7, P>0.05). Thus, reduced sAPPα secretion by BACE2siRNA treatment is likely caused not only by decreased α-secretase-like function of BACE2, but also by reduced APP expression. We further examined the effects of BACE2siRNA in senescent human BMECs. In cultured human BMECs (passage 22), protein expressions of senescent markers (p 21Cip1 and p 16INK4a ) were significantly increased (n=4, P<0.05). Genetic inactivation of BACE2 in senescent human BMECs also significantly suppressed secretion of sAPPα (n=8, P<0.05), but did not affect Aβ40 production (n=8, P>0.05). BACE2-siRNA treatment significantly inhibited protein expressions of APP and mature ADAM10 (n=7, P<0.05), but did not change BACE1 protein expression (n=7, P>0.05). Thus in senescent human BMECs, reduced APP expression and impaired α-processing may play important roles in the decreased sAPPα production. Since our previous studies have demonstrated that endothelial production of sAPPα significantly contributes to the sAPPα content in the hippocampus, our current findings suggests that inhibition of BACE2 could impair protective function of sAPPα in the hippocampus.

The role of oxidative stress and antioxidant enzymes in the aging related dysfunction of EPCs has not been studied. We hypothesized that impaired antioxidant capacity contributes to dysfunction of EPCs. Early EPCs isolated from healthy young (24±3 year‐old, n=9) and aged (71±5 year‐old, n=9) males were examined. The levels of protein expression and enzymatic activity of glutathione peroxidase 1 (GPX1) were decreased significantly in the EPCs from aged individuals in compared to EPCs from young individuals (n=9, P<0.05). After 24 h of serum starvation EPC numbers in young group and aged group were 0.6±0.2 and 0.4±0.1, respectively (expressed as ratio to the cell number under normal culture conditions, n=6, P<0.05). H 2 O 2 (1mM) treatment for 24 h significantly decreased EPC number to 37±13% in aged, compared with young individuals (67±13%, n=3, P<0.05). Furthermore, under normal culture conditions the numbers of senescent β‐galactosidase (β‐gal) positive EPCs were not significantly different between young and aged subjects (46.1±8.4%, and 58.3±7.6, respectively, n=3). However, H 2 O 2 (50 μM) treatment significantly increased the β‐gal positive cells in aged EPCs (73±9%), compared with young EPCs (43±11%, n=3, P<0.05). These results suggest that EPCs of older individuals are more susceptible to oxidative stress induced by H 2 O 2 and serum starvation, resulting in decreased survival and increased senescence. Reduced expression of GPX1 may contribute to EPC dysfunction with aging.

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 regenerative function of endothelial progenitor cells (EPCs) are not completely understood. Since previous studies from several groups including ours suggest that biosynthesis of NO in EPCs is low, the present study was designed to determine ability of EPCs to produce and release prostacyclin (PGI 2 ). Outgrowth (“late”) EPCs were obtained from peripheral blood of healthy subjects. Enzymatic activity of eNOS was significantly lower in EPCs as compared to eNOS activity in human coronary endothelial cells (CAECs) (n=4‐6 subjects, P<0.05). In contrast, production and release of PGI 2 was four fold higher in EPCs as compared to CAECs (n=3, P<0.05). This was associated with significantly higher expression of cyclooxygenase‐1 (COX‐1) protein in EPCs whereas levels of COX‐2 protein were not different between progenitor and mature endothelial cells. Production of thromboxane A 2 and prostaglandin E 2 were low and identical in EPCs and CAECs. Most importantly, inhibition of COX isoforms with indomethacin impaired pro‐angiogenic effect of EPCs as assessed by in vitro tube formation assay (n=4‐6, P<0.05). Cell proliferation was also significantly reduced after inhibition of COX isoforms with indomethacin or inactivation of PGI 2 ‐synthase by siRNA (n=4‐6, P<0.05). Our findings suggest that production and release PGI 2 is an important mechanism contributing to regenerative capability of EPCs.

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.

The exact physiological function of amyloid-β precursor protein (APP) in endothelial cells is unknown. Endothelium-specific APP-deficient (eAPP −/− ) mice were created to gain new insights into the role of APP in the control of vascular endothelial function. Endothelium-dependent relaxations to acetylcholine were significantly impaired in basilar arteries of global APP knockout (APP −/− ) and eAPP −/− mice ( P < 0.05). In contrast, endothelium-independent relaxations to nitric oxide (NO)-donor diethylamine-NONOate were unchanged. Western blot analysis revealed that protein expression of endothelial nitric oxide synthase (eNOS) was significantly downregulated in large cerebral arteries of APP −/− mice and eAPP −/− mice as compared to respective wild-type littermates ( P < 0.05). Furthermore, basal levels of cyclic guanosine monophosphate (cGMP) were also significantly reduced in large cerebral arteries of APP-deficient mice ( P < 0.05). In contrast, protein expression of prostacyclin synthase as well as levels of cyclic adenosine monophosphate (cAMP) was not affected by genetic inactivation of APP in endothelial cells. By using siRNA to knockdown APP in cultured human brain microvascular endothelial cells we also found a significant downregulation of eNOS mRNA and protein expressions in APP-deficient endothelium ( P < 0.05). These findings indicate that under physiological conditions, expression of APP in cerebral vascular endothelium plays an important protective function by maintaining constitutive expression of eNOS .