Cerebrovascular Endothelial Dysfunction: Role of BACE1.

Dysfunctional endothelium is increasingly recognized as a mechanistic link between cardiovascular risk factors and dementia, including Alzheimer disease. BACE1 (ß-site amyloid-ß precursor protein-cleaving enzyme 1) is responsible for ß-processing of APP (amyloid-ß precursor protein), the first step in the production of Aß (amyloid-ß) peptides, major culprits in the pathogenesis of Alzheimer disease. Under pathological conditions, excessive activation of BACE1 exerts detrimental effects on endothelial function by Aß-dependent and Aß-independent mechanisms. High local concentration of Aß in the brain blood vessels is responsible for the loss of key vascular protective functions of endothelial cells. More recent studies recognized significant contribution of Aß-independent proteolytic activity of endothelial BACE1 to the pathogenesis of endothelial dysfunction. This review critically evaluates existing evidence supporting the concept that excessive activation of BACE1 expressed in the cerebrovascular endothelium impairs key homeostatic functions of the brain blood vessels. This concept has important therapeutic implications. Indeed, improved understanding of the mechanisms of endothelial dysfunction may help in efforts to develop new approaches to the protection and preservation of healthy cerebrovascular function.

Emerging Roles of Endothelial Nitric Oxide in Preservation of Cognitive Health.

eNOS (endothelial nitric oxide synthase) is critically important enzyme responsible for regulation of cardiovascular homeostasis. Under physiological conditions, constitutive eNOS activity and production of endothelial nitric oxide (NO) exert essential neurovascular protective functions. In this review, we first discuss the roles of endothelial NO in prevention of neuronal amyloid accumulation and formation of neurofibrillary tangles, hallmarks of Alzheimer disease pathology. Next, we review existing evidence suggesting that NO released from endothelium prevents activation of microglia, stimulates glycolysis in astrocytes, and increases biogenesis of mitochondria. We also address major risk factors for cognitive impairment including aging and ApoE4 (apolipoprotein 4) genotype with focus on their detrimental effects on eNOS/NO signaling. Relevant to this review, recent studies suggested that aged eNOS heterozygous mice are unique model of spontaneous cerebral small vessel disease. In this regard, we review contribution of dysfunctional eNOS to deposition of Aß (amyloid-ß) into blood vessel wall leading to development of cerebral amyloid angiopathy. We conclude that endothelial dysfunction manifested by the loss of neurovascular protective functions of NO may significantly contribute to development of cognitive impairment.

BACE2 deficiency impairs expression and function of endothelial nitric oxide synthase in brain endothelial cells.

Beta-site amyloid precursor protein (APP)-cleaving enzyme 2 (BACE2) is highly expressed in cerebrovascular endothelium. Notably, BACE2 is one of the most downregulated genes in cerebrovascular endothelium derived from patients with Alzheimer's disease. The present study was designed to determine the role of BACE2 in control of expression and function of endothelial nitric oxide synthase (eNOS). Genetic downregulation of BACE2 with small interfering RNA (BACE2siRNA) in human brain microvascular endothelial cells (BMECs) significantly decreased expression of eNOS and elevated levels of eNOS phosphorylated at threonine residue Thr495, thus leading to reduced production of nitric oxide (NO). BACE2siRNA also suppressed expression of APP and decreased production and release of soluble APPα (sAPPα). In contrast, adenovirus-mediated overexpression of APP increased expression of eNOS. Consistent with these observations, nanomolar concentrations of sAPPα and APP 17mer peptide (derived from sAPPα) augmented eNOS expression. Further analysis established that γ-aminobutyric acid type B receptor subunit 1 and Krüppel-like factor 2 may function as downstream molecular targets significantly contributing to BACE2/APP/sAPPα-induced up-regulation of eNOS. In agreement with studies on cultured human endothelium, endothelium-dependent relaxations to acetylcholine and basal production of cyclic GMP were impaired in cerebral arteries of BACE2-deficient mice. We propose that in the brain blood vessels, BACE2 may function as a vascular protective protein.

Expression and Processing of Amyloid Precursor Protein in Vascular Endothelium.

Amyloid precursor protein (APP) is evolutionary conserved protein expressed in endothelial cells of cerebral and peripheral arteries. In this review, we discuss mechanisms responsible for expression and proteolytic cleavage of APP in endothelial cells. We focus on physiological and pathological implications of APP expression in vascular endothelium.

Uncoupling of endothelial nitric oxide synthase in cerebral vasculature of Tg2576 mice.

In this study, we tested the hypothesis that reduced bioavailability of tetrahydrobiopterin (BH4) is a major mechanism responsible for pathogenesis of endothelial dysfunction in cerebral microvessels of transgenic mice expressing the Swedish double mutation of human amyloid precursor protein (APP) (Tg2576 mice). Endothelial nitric oxide synthase (eNOS) protein expression was significantly increased in cerebral vasculature of Tg2576 mice. In contrast, bioavailability of BH4 was significantly reduced (p < 0.05). Moreover, superoxide anion production was increased in cerebral microvessels of Tg2576 mice (p < 0.05). Incubation with NOS inhibitor, Nω-nitro-L-arginine methyl ester, decreased superoxide anion indicating that uncoupled eNOS is most likely the source of superoxide anion. Increasing BH4 bioavailability either exogenously by BH4 supplementation or endogenously by treatment with the selective peroxisome proliferator-activated receptor--delta activator GW501516 (2 mg/kg/day, 14 days) attenuated eNOS uncoupling and decreased superoxide anion production in cerebral microvessels of Tg2576 mice (p < 0.05). Treatment with GW501516 restored the biological activity of endothelial nitric oxide in cerebral microvessels of Tg2576 mice, as indicated by the increased nitrite/nitrate content and 3,5-cyclic guanosine monophosphate levels (p < 0.05). Our studies indicate that sub-optimal BH4 bioavailability in cerebral vasculature is an important contributor to oxidant stress and endothelial dysfunction in Tg2576 mouse model of Alzheimer's disease. Existing evidence suggests that Aß peptides-induced up-regulation of expression and activity of NADPH oxidase causes increased production of superoxide anion (.O2(-)). .O2(-) can also be converted to hydrogen peroxide (H2O2) by enzymatic activity of superoxide dismutase (SOD) or spontaneous dismutation. Elevation of .O2(-) and H2O2 might cause oxidation of tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2) and subsequent uncoupling of endothelial nitric oxide synthase (eNOS) (a) thus reducing levels of nitric oxide (NO) and 3',5'-cyclic guanosine monophosphate (cGMP). Supplementation of BH4 or activation of PPARδ prevents detrimental effects of eNOS uncoupling by restoring bioavailability of BH4 and scavenging of .O2(-), respectively (b). Activation of PPARδ also increases expression of catalase thereby inactivating H2O2. Generation of H2O2 by uncoupled eNOS in cerebral microvessels of Tg2576 mice is hypothetical.

Mechanisms of vascular dysfunction in mice with endothelium-specific deletion of the PPAR-δ gene.

Peroxisome proliferator-activated receptor (PPAR)-δ is a nuclear hormone receptor that is mainly involved in lipid metabolism. Recent studies have suggested that PPAR-δ agonists exert vascular protective effects. The present study was designed to characterize vascular function in mice with genetic inactivation of PPAR-δ in the endothelium. Mice with vascular endothelial cell-specific deletion of the PPAR-δ gene (ePPARδ(-/-) mice) were generated using loxP/Cre technology. ePPARδ(-/-) mice were normotensive and did not display any sign of metabolic syndrome. Endothelium-dependent relaxations to ACh and endothelium-independent relaxations to the nitric oxide (NO) donor diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate were both significantly impaired in the aorta and carotid arteries of ePPARδ(-/-) mice (P < 0.05). In ePPARδ(-/-) mouse aortas, phosphorylation of endothelial NO synthase at Ser(1177) was significantly decreased (P < 0.05). However, basal levels of cGMP were unexpectedly increased (P < 0.05). Enzymatic activity of GTP-cyclohydrolase I and tetrahydrobiopterin levels were also enhanced in ePPARδ(-/-) mice (P < 0.05). Most notably, endothelium-specific deletion of the PPAR-δ gene significantly decreased protein expressions of catalase and glutathione peroxidase 1 and resulted in increased levels of H2O2 in the aorta (P < 0.05). In contrast, superoxide anion production was unaltered. Moreover, treatment with catalase prevented the endothelial dysfunction and elevation of cGMP detected in aortas of ePPARδ(-/-) mice. The findings suggest that increased levels of cGMP caused by H2O2 impair vasodilator reactivity to endogenous and exogenous NO. We speculate that chronic elevation of H2O2 predisposes PPAR-δ-deficient arteries to oxidative stress and vascular dysfunction.

Brain-derived neurotrophic factor increases expression of MnSOD in human circulating angiogenic cells.

Existing evidence suggests that brain-derived neurotrophic factor (BDNF) promotes survival and proliferation of endothelial cells, stimulates mobilization of hematopoietic progenitors, and induces angiogenesis in ischemic tissues. However, the mechanisms underlying vascular protective function of BDNF are poorly understood. We hypothesized that BDNF increases antioxidant capacity of circulating angiogenic cells. Human mononuclear cells were isolated from peripheral blood of 30 healthy male volunteers (48±2 years old), and cultured in endothelial growth medium-2 for 4-5 days. The attached cells (so called early endothelial progenitor cells [early EPCs], or circulating angiogenic cells) expressed BDNF receptors, tropomyosin-related kinase B and p75 neurotrophin receptor. Treatment of early EPCs with recombinant human BDNF for 24 h significantly increased manganese superoxide dismutase (MnSOD) expression, but had no effect on expression of other antioxidant enzymes including copper zinc SOD (CuZnSOD), catalase, and glutathione peroxidase-1. BDNF stimulated phosphorylation of IκB kinase (IKK)α/ß and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK); however it did not activate p38, Erk, or AKT. Treatment with nuclear factor κB inhibitor, PDTC, or JNK inhibitor, SP600125, attenuated BDNF-augmented MnSOD protein expression. BDNF treatment inhibited apoptosis induced by a superoxide anion generator LY83583, and serum starvation-induced cell detachment. These findings suggest that BDNF protects EPCs by increasing expression of MnSOD thereby enhancing their antioxidant capacity.

Muscle-specific f-box only proteins facilitate bk channel ß(1) subunit downregulation in vascular smooth muscle cells of diabetes mellitus.

RATIONALE: activity of the large conductance Ca(2+)-activated K(+) (BK) channels is profoundly modulated by its ß(1) subunit (BK-ß(1)). However, BK-ß(1) expression is downregulated in diabetic vessels. The ubiquitin-proteasome system (UPS) is a major mechanism of intracellular protein degradation. Whether UPS participates in BK-ß(1) downregulation in diabetic vessels is unknown. OBJECTIVE: we hypothesize that UPS facilitates vascular BK-ß(1) degradation in diabetes. METHODS AND RESULTS: using patch clamp and molecular biological approaches, we found that BK-ß(1)-mediated channel activation and BK-ß(1) protein expression were reduced in aortas of streptozotocin-induced diabetic rats and in human coronary arterial smooth muscle cells (CASMCs) cultured in high glucose. This was accompanied by upregulation of F-box only protein (FBXO)-9 and FBXO-32 (atrogin-1), the key components of the Skp1-Cullin-F-box (SCF) type ubiquitin ligase complex. BK-ß(1) expression was suppressed by the FBXO activator doxorubicin but enhanced by FBXO-9 small interfering RNA or by the proteasome inhibitor MG-132. Cotransfection of atrogin-1 in HEK293 cells significantly reduced Flag-hSlo-ß(1) expression by 2.16-fold, compared with expression of Flag-hSlo-ß(1)V146A (a mutant without the PDZ-binding motif). After cotransfection with atrogin-1, the ubiquitination of Flag-hSlo-ß(1) was increased by 1.91-fold, compared with that of hSlo-ß(1)V146A, whereas cotransfection with atrogin-1ΔF (a nonfunctional mutant without the F-box motif) had no effect. Moreover, inhibition of Akt signaling attenuated the phosphorylation of forkhead box O transcription factor (FOXO)-3a and enhanced atrogin-1 expression, which in turn suppressed BK-ß(1) protein levels in human CASMCs. CONCLUSIONS: downregulation of vascular BK-ß(1) expression in diabetes and in high-glucose culture conditions was associated with FOXO-3a/FBXO-dependent increase in BK-ß(1) degradation.

Regulation of coronary arterial BK channels by caveolae-mediated angiotensin II signaling in diabetes mellitus.

RATIONALE: The large conductance Ca(2+)-activated K(+) (BK) channel, a key determinant of vascular tone, is regulated by angiotensin II (Ang II) type 1 receptor signaling. Upregulation of Ang II functions and downregulation of BK channel activities have been reported in diabetic vessels. However, the molecular mechanisms underlying Ang II-mediated BK channel modulation, especially in diabetes mellitus, have not been thoroughly examined. OBJECTIVES: The aim in this study was to determine whether caveolae-targeting facilitates BK channel dysfunction in diabetic vessels. METHODS AND RESULTS: Using patch clamp techniques and molecular biological approaches, we found that BK channels, Ang II type 1 receptor, G(alphaq/11) (G protein q/11 alpha subunit), nonphagocytic NAD(P)H oxidases (NOX-1), and c-Src kinases (c-Src) were colocalized in the caveolae of rat arterial smooth muscle cells and the integrity of caveolae in smooth muscle cells was critical for Ang II-mediated BK channel regulation. Most importantly, membrane microdomain targeting of these proteins was upregulated in the caveolae of streptozotocin-induced rat diabetic vessels, leading to enhanced Ang II-induced redox-mediated BK channel modification and causing BK channel and coronary dysfunction. The absence of caveolae abolished the effects of Ang II on vascular BK channel activity and preserved BK channel function in diabetes. CONCLUSIONS: These results identified a molecular scheme of receptor/enzyme/channel/caveolae microdomain complex that facilitates the development of vascular BK channel dysfunction in diabetes.

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

Cerebrovascular effects of ß-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 ß-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.

Angiogenic function of prostacyclin biosynthesis in human endothelial progenitor cells.

The role of prostaglandin production in the control of regenerative function of endothelial progenitor cells (EPCs) has not been studied. We hypothesized that activation of cyclooxygenase (COX) enzymatic activity and the subsequent production of prostacyclin (PGI(2)) is an important mechanism responsible for the regenerative function of EPCs. In the present study, we detected high levels of COX-1 protein expression and PGI(2) biosynthesis in human EPCs outgrown from blood mononuclear cells. Expression of COX-2 protein was almost undetectable under basal conditions but significantly elevated after treatment with tumor necrosis factor-alpha. Condition medium derived from EPCs hyperpolarized human coronary artery smooth muscle cells, similar to the effect of the PGI(2) analog iloprost. The proliferation and in vitro tube formation by EPCs were inhibited by the COX inhibitor indomethacin or by genetic inactivation of COX-1 or PGI(2) synthase with small interfering (si)RNA. Impaired tube formation and cell proliferation induced by inactivation of COX-1 were rescued by the treatment with iloprost or the selective peroxisome proliferator-activated receptor (PPAR)delta agonist GW501516 but not by the selective PGI(2) receptor agonist cicaprost. Downregulation of PPARdelta by siRNA also reduced angiogenic capacity of EPCs. Iloprost failed to reverse PPARdelta siRNA-induced impairment of angiogenesis. Furthermore, transfection of PGI(2) synthase siRNA, COX-1 siRNA, or PPARdelta siRNA into EPCs decreased the capillary formation in vivo after transplantation of human EPCs into the nude mice. These results suggest that activation of COX-1/PGI(2)/PPARdelta pathway is an important mechanism underlying proangiogenic function of EPCs.

Aging decreases expression and activity of glutathione peroxidase-1 in human endothelial progenitor cells.

The mechanisms underlying effects of aging on functions of pro-angiogenic endothelial progenitor cells (EPCs) are poorly understood. Previous studies demonstrated that human EPCs express high levels of antioxidant enzymes as compared to mature endothelial cells. Here, we hypothesized that aging impairs antioxidant capacity of EPCs. So called "early EPCs" derived from cultured blood mononuclear cells were obtained from healthy young (average=24 years old) and old (average=72 years old) subjects. In EPCs of old subjects, the levels of glutathione peroxidase-1 (GPX1) protein and enzymatic activity were significantly reduced. The serum selenium levels in young and old subjects were not significantly different. Increasing selenium concentration in the cell culture also did not affect the protein levels of GPX1, suggesting the reduced GPX1 in old subject's EPCs is selenium independent. Expressions of catalase, Mn-superoxide dismutase (MnSOD), and CuZnSOD were not affected by aging. EPCs of old subjects were more sensitive to oxidative stress induced by H(2)O(2) as compared with EPCs of young subjects, suggesting that impairment of GPX1 during aging may contribute to low survival ability of EPCs in response to oxidative stress. The results indicate that GPX1 may represent a potential therapeutic target for enhancement of regenerative capacity of EPCs in old subjects.

Endothelial progenitor cells stimulate cerebrovascular production of prostacyclin by paracrine activation of cyclooxygenase-2.

In the present study we hypothesized that endothelial progenitor cells (EPCs) enhance production of vasoprotective substances in cerebral arteries. Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium (EGM-2) for 7 days to yield EPCs. Rabbit basilar arteries were exposed to autologous EPCs ( approximately 5x10(5) cells) in vitro or in vivo. Twenty-four hours after intracisternal delivery of autologous EPCs, basilar arteries were isolated and expression of vasoregulatory proteins, production of prostacyclin (PGI(2)), and cAMP were determined. Arteries transplanted with EPCs demonstrated increased protein expression of cyclooxygenase-2 and PGI(2) in adventitia, media, and endothelium. Furthermore, production of PGI(2) and arterial content of cAMP, second messenger for PGI(2), were significantly augmented after transplantation of EPCs. In contrast, production of thromboxane A(2) was significantly reduced, whereas production of prostaglandin E(2) remained unchanged. The increased production of PGI(2) and arterial content of cAMP were inhibited only by a selective cyclooxygenase-2 inhibitor, NS-398. In vitro or in vivo treatment of basilar artery with conditioned media from EPCs also caused increase in cyclooxygenase-2 and PGI(2) synthase protein expression associated with elevation of cAMP. Our results suggest that in cerebral arteries, paracrine effect of EPCs promotes vasoprotection by increasing PGI(2) production and intracellular concentration of cAMP. This effect appears to be mediated by activation of arachidonic acid metabolism via stimulation of cyclooxygenase-2/PGI(2) synthase pathway.

Impairment of amyloid precursor protein alpha-processing in cerebral microvessels of type 1 diabetic mice.

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 A2 (TXA2). In vitro studies in human brain microvascular endothelial cells (BMECs) revealed that knockdown of AC3 significantly suppressed ADAM10 protein levels, and that activation of TXA2 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.

Impaired Ca2+-dependent activation of large-conductance Ca2+-activated K+ channels in the coronary artery smooth muscle cells of Zucker Diabetic Fatty rats.

The large-conductance Ca(2+)-activated K(+) (BK) channels play an important role in the regulation of cellular excitability in response to changes in intracellular metabolic state and Ca(2+) homeostasis. In vascular smooth muscle, BK channels are key determinants of vasoreactivity and vital-organ perfusion. Vascular BK channel functions are impaired in diabetes mellitus, but the mechanisms underlying such changes have not been examined in detail. We examined and compared the activities and kinetics of BK channels in coronary arterial smooth muscle cells from Lean control and Zucker Diabetic Fatty (ZDF) rats, using single-channel recording techniques. We found that BK channels in ZDF rats have impaired Ca(2+) sensitivity, including an increased free Ca(2+) concentration at half-maximal effect on channel activation, a reduced steepness of Ca(2+) dose-dependent curve, altered Ca(2+)-dependent gating properties with decreased maximal open probability, and a shortened mean open-time and prolonged mean closed-time durations. In addition, the BK channel beta-subunit-mediated activation by dehydrosoyasaponin-1 (DHS-1) was lost in cells from ZDF rats. Immunoblotting analysis confirmed a 2.1-fold decrease in BK channel beta(1)-subunit expression in ZDF rats, compared with that of Lean rats. These abnormalities in BK channel gating lead to an increase in the energy barrier for channel activation, and may contribute to the development of vascular dysfunction and complications in type 2 diabetes mellitus.

Molecular mechanisms mediating inhibition of human large conductance Ca2+-activated K+ channels by high glucose.

Diabetic vascular dysfunction is associated with an increase in reactive oxygen species (ROS). In this study, we hypothesized that hyperglycemia-induced ROS generation would impair the function of large conductance Ca(2+)-activated K(+) (BK) channels, which are major determinants in vasorelaxation. We found that when cultured in high glucose (HG) (22 mmol/L), HEK293 cells showed a reduction in expressed hSlo current densities, as well as slowed activation and deactivation kinetics. When human coronary smooth muscle cells were cultured in HG, similar findings were observed for the BK currents. HG enhanced superoxide dismutase and suppressed catalase (CAT) expression in HEK293 cells, leading to a significant increase in intracellular ROS. The effects of HG were mimicked by hydrogen peroxide (H(2)O(2)), and hSlo functions were restored by CAT gene transfer. Peroxynitrite inhibited hSlo current density but did not change channel kinetics. The hSloC911A mutant was insensitive to the effects of HG and H(2)O(2). Hence, imbalance of antioxidant enzymes plays a critical role in ROS generation in HG, impairing hSlo functions through H(2)O(2)-dependent oxidation at cysteine 911. This may represent an important fundamental mechanism that contributes to the impairment of vasodilation in diabetes.

Effects of Brain-Derived Neurotrophic Factor on MicroRNA Expression Profile in Human Endothelial Progenitor Cells.

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.

Effects of senescence and angiotensin II on expression and processing of amyloid precursor protein in human cerebral microvascular endothelial cells.

The present study was designed to determine the effects of senescence and angiotensin II (Ang II) on expression and processing of amyloid precursor protein (APP) in human brain microvascular endothelial cells (BMECs). Senescence caused a decrease in APP expression thereby resulting in reduced secretion of soluble APPα (sAPPα). In contrast, ß-site APP cleaving enzyme (BACE1) expression and production of amyloid ß (Aß)40 were increased in senescent endothelium. Importantly, in senescent human BMECs, treatment with BACE1 inhibitor IV inhibited Aß generation and increased sAPPα production by enhancing a disintegrin and metalloprotease (ADAM)10 expression. Furthermore, Ang II impaired expression of ADAM10 and significantly reduced generation of sAPPα in senescent human BMECs. This inhibitory effect of Ang II was prevented by treatment with BACE1 inhibitor IV. Our results suggest that impairment of α-processing and shift to amyloidogenic pathway of APP contribute to endothelial dysfunction induced by senescence. Loss of sAPPα in senescent cells treated with Ang II exacerbates detrimental effects of senescence on APP processing. Notably, inhibition of BACE1 has beneficial effects on senescence induced endothelial dysfunction. Reported findings may help to explain contributions of senescent cerebral microvascular endothelium to development of cerebral amyloid angiopathy and Alzheimer's disease (AD) pathology.

Endothelium-specific amyloid precursor protein deficiency causes endothelial dysfunction in cerebral arteries.

The exact physiological function of amyloid-ß 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 .

Role of prostacyclin signaling in endothelial production of soluble amyloid precursor protein-α in cerebral microvessels.

We tested hypothesis that activation of the prostacyclin (PGI2) receptor (IP receptor) signaling pathway in cerebral microvessels plays an important role in the metabolism of amyloid precursor protein (APP). In human brain microvascular endothelial cells activation of IP receptor with the stable analogue of PGI2, iloprost, stimulated expression of amyloid precursor protein and a disintegrin and metalloprotease 10 (ADAM10), resulting in an increased production of the neuroprotective and anticoagulant molecule, soluble APPα (sAPPα). Selective agonist of IP receptor, cicaprost, and adenylyl cyclase activator, forskolin, also enhanced expression of amyloid precursor protein and ADAM10. Notably, in cerebral microvessels of IP receptor knockout mice, protein levels of APP and ADAM10 were reduced. In addition, iloprost increased protein levels of peroxisome proliferator-activated receptor δ (PPARδ) in human brain microvascular endothelial cells. PPARδ-siRNA abolished iloprost-augmented protein expression of ADAM10. In contrast, GW501516 (a selective agonist of PPARδ) upregulated ADAM10 and increased production of sAPPα. Genetic deletion of endothelial PPARδ (ePPARδ-/-) in mice significantly reduced cerebral microvascular expression of ADAM10 and production of sAPPα. In vivo treatment with GW501516 increased sAPPα content in hippocampus of wild type mice but not in hippocampus of ePPARδ-/- mice. Our findings identified previously unrecognized role of IP-PPARδ signal transduction pathway in the production of sAPPα in cerebral microvasculature.