He, Tongrong

Aged heterozygous endothelial nitric oxide synthase knockout mice (eNOS+/−) represent an important model of spontaneous cerebral small vessel disease. However, the role of endothelium in vasomotor function of cerebral arteries of aged eNOS+/− mice has not been characterized. In the process of generating endothelial specific β-site amyloid precursor protein cleaving enzyme-1 (BACE1)/eNOS+/− knockout mice, we performed control studies in 18 months old male eNOS+/− mice and their wild-type littermates generated on floxed C57BL/6-Bace1tm1.1mrl background. Vasomotor function of isolated basilar arteries was studied in vitro under pressurized conditions using a video dimensions analyzer. Contractions to acetylcholine were observed in aged eNOS+/− mice (19±13%), while acetylcholine caused endothelium-dependent relaxations in age-matched wild-type littermates (-17±7%, P< 0.05; n=9 per group). In contrast, endothelium-independent relaxations to NO-donor diethylamine-NONOate were unaltered in both wild-type littermates (-83±10%) and eNOS+/− mice (-90±4%; n=6 per group; P >0.05). Furthermore, contractions to U46619 were significantly augmented in eNOS+/− mice (63±3%; P< 0.05 vs. wild-type: 47±4%; n=6). Morphological analysis revealed that lumen diameter of basilar arteries was not different between wild-type (169±4 µm) and eNOS+/− mice (165±4 µm; P >0.05; n=9). Furthermore, blood glucose levels were comparable between wild-type (185±7 mg/dL) and eNOS+/− mice (183±12 mg/dL; P >0.05; n=10). Measurement of lipid profile showed that plasma cholesterol, HDL as well as triglyceride levels were unchanged in eNOS+/− mice (P >0.05 vs. wild-type; n=10). Interestingly, mRNA expression of BACE1 is significantly increased in cerebral arteries of aged eNOS+/− mice (0.0295±0.0009) as compared to wild-type littermates (0.0243±0.0017; P< 0.05; n=5 per group). Our results demonstrate that heterozygous eNOS deficiency exacerbates endothelial dysfunction in aged eNOS+/− mice and is accompanied by the imbalance between vasodilation and vasoconstriction of cerebral arteries. Endothelial dysfunction in aged eNOS+/− mice appears to be independent of alterations in systemic metabolic parameters. Notably, partial deficiency of eNOS increases expression of BACE1, an enzyme responsible for production of amyloid-β peptides. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Beta-site amyloid precursor protein cleaving enzyme 2 (BACE2) is one of the most downregulated genes in the brain capillary endothelial cells derived from patients with Alzheimer's disease (AD). Endothelin-1 (ET-1) significantly contributes to the pathogenesis of AD. We hypothesized that loss of BACE2 increases production of ET-1 from human brain microvascular endothelial cells (BMECs). Genetic inactivation of BACE2 in cultured human BMECs significantly upregulated expression and release of ET-1. Mechanistic studies indicated that γ-aminobutyric acid type B receptor subunit 2/transforming growth factor beta 2 signaling pathway mediated the effect of BACE2 inhibition on ET-1 production.

The exact physiological role of BACE2 in endothelial function of systemic arteries is unknown. Our study shows that thromboxane A2 and prostaglandin F2α are responsible for impairment of endothelium-dependent relaxations in endothelium-specific BACE2-deficient mice. The data support an important role of BACE2 in the control of endothelial arachidonic acid metabolism and vasomotor function.

Dysfunctional endothelium is increasingly recognized as a mechanistic link between cardiovascular risk factors and dementia, including Alzheimer disease. BACE1 (β-site amyloid-β precursor protein-cleaving enzyme 1) is responsible for β-processing of APP (amyloid-β precursor protein), the first step in the production of Aβ (amyloid-β) peptides, major culprits in the pathogenesis of Alzheimer disease. Under pathological conditions, excessive activation of BACE1 exerts detrimental effects on endothelial function by Aβ-dependent and Aβ-independent mechanisms. High local concentration of Aβ 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 Aβ-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.

Background: We have found that the vascular large conductance Ca 2+ -activated K + channels (BK) are targeted to caveolae. The goal of this study is to determine the role of caveolae in BK regulation by angiotensin II (AII) in normal and diabetic rats. Results: In coronary arterial smooth muscle cells (CASMC) from control rats, AII (2 μ M) inhibited BK by 40.5% from 227±26 pA/pF to 135±14 pA/pF (HP −60 mV, TP 150 mV, n=7, p<0.05). However, AII had no effects on BK in CASMC transfected with caveolin-1 siRNA (vs 50% inhibition in cells transfected with control siRNA) or in cells from Cav1−/− mice, suggesting the AII effects are caveolae-dependent. Density gradient analysis of rat vessel homogenates showed that caveolin-1, BK, Gq, cSrc, and NOX-1 all reside in the caveolae-rich fraction, where AT 1 R was absent unless the animal was pre-treated with AII, suggesting receptor translocation upon agonist activation. However, CASMC from streptozotocin-induced diabetic rats showed significant reduction in BK (61.5±10.6 pA/pF) with no response to AII (61.2±15.7 pA/pF, n=8). Vascular BK, caveolin-1, AT 1 R, cSrc, and NOX-1 expression in caveolae were all upregulated by 1.6- to 3.1-fold in diabetes. Density gradient analysis of diabetic vessels showed the presence of AT 1 R in the caveolae-rich fraction without AII pretreatment, suggesting that the AT 1 R signaling pathway might be constitutively active in diabetes. This was accompanied by a 2-fold increase in BK tyrosine phosphorylation and BK tyrosine nitration. The AII effects on BK function in diabetic CASMC were abolished by pretreatment (1 hr) with PKC inhibitor or by LavA (cSrc inhibitor) and can be mimicked by incubation (2 hr) of control CASMC with peroxynitrite. Conclusion: AT 1 R-BK-enzyme complex in the caveolae microdomain is critical in vascular BK regulation. Enhanced AII-signaling facilitates BK post-translational modification, resulting in BK dysfunction in diabetes.

AbstractBeta‐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.image

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 Aβ (amyloid-β) 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.

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.

In the present study, we tested hypothesis that hyperglycemia impairs the neuro-protective function of cerebral microvessels. Type 1 diabetes (T1D) was induced by treatment of mice with streptozotocin (blood glucose >300 mg/dL for 2 months). Brain tissues and cerebral microvessels were isolated from diabetic and control mice. We observed significant decrease in the protein levels of soluble amyloid precursor protein-α (sAPPα, a product of α-cleavage of APP and a neurotrophic and neuroprotective molecule) in the total brain tissue of diabetic mice (n=5, P<0.05). Further examination of brain regions revealed that the reduction of protein levels of sAPPα was only detected in hippocampus and cortex but not in cerebellum (n=6, P<0.05). Our studies also revealed that there were no significant differences in the protein expression of APP and its non-amyloidogenic processing enzymes, α-secretases (a disintegrin and metalloprotease 10 [ADAM10], ADAM9, and ADAM17) in total brain tissue between T1D mice and control mice (n=5-6, P>0.05). The protein level of β-processing enzyme BACE1 was also not changed in T1D mouse brain. In contrast, the protein levels of APP and ADAM10 were significantly decreased in cerebral microvasculature of T1D mice, as compared to control mice (n=9-10, P<0.05). Immunofluorescent confocal microscopy also demonstrated that expressions of APP and ADAM10 in cerebral microvasculature (identified by co-staining with CD31) in hippocampus and cortex were reduced in T1D mice. Of note, cerebrovascular protein levels of ADAM9, ADAM17 and BACE1 were not affected by hyperglycemia (n=5-7, P>0.05). These results suggest that hyperglycemia impairs APP expression and α-processing in brain vasculature, thereby leading to the reduction of brain content of sAPPα. The selective loss of neuro-protective molecule sAPPα in hippocampus and cortex may be an important mechanism contributing to the development of cognitive dysfunction in diabetes.

We tested the hypothesis that endothelial PPARδ plays an important role in the protection of neurovascular unit. Cerebral microvessels were isolated from mice with endothelium-specific deficiency of PPARδ (ePPARδ-/-) and littermate wild type (WT) mice. After ex vivo incubation of cerebral microvessels for 24 h, supernatant was collected and levels of soluble amyloid precursor protein α [sAPPα, a product of non-amyloidogenic processing of amyloid precursor protein (APP)] were measured using an ELISA kit. The production of sAPPα in cerebral microvessels of ePPARδ-/- mice was reduced by 54%, as compared to WT mice (n=6-7, P<0.05). Western blot analysis demonstrated that microvascular expression of ADAM10 was reduced in ePPARδ-/- mice (80%) (n=18, P<0.05), while ADAM17 protein level was increased in by 132% (n=4, P<0.05), as compared to WT mice. Protein expressions of APP, ADAM9, and BACE1 were not significantly different between cerebral microvessels derived from ePPARδ-/- mice and WT mice. Real-time RT-PCR demonstrated that in brain microvessels of ePPARδ-/- mice mRNA levels of ADAM10 were also decreased by 56% (n=5, P<0.05). Furthermore, Western blot analysis of the brain regions showed that sAPPα content was selectively reduced in hippocampus of ePPARδ-/- mice by 80% (n=11, P<0.05). This was associated with significantly reduced expression of ADAM10 in the hippocampus (n=11, P<0.05). In contrast, protein levels of ADAM10, and sAPPα were not altered in cortex and cerebellum of ePPARδ-/- mice. In addition, we did not detect any changes in Aβ40 and Aβ42 levels (measured using ELISA assay) in all three examined brain regions. Since sAPPα is a potent neurotrophic and neuro-protective molecule, our results suggest that loss of endothelial PPARδ may selectively increase vulnerability of hippocampus. This mechanism may contribute to susceptibility of hippocampus to injury during initiation and progression of neuro-degenerative diseases.