Citations

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.

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.

Abstract Aim We aimed to test the hypothesis that a high‐salt diet (HS) impairs NO signaling in kidney microvascular endothelial cells through a histone deacetylase 1 (HDAC1)‐dependent mechanism. Methods Male Sprague Dawley rats were fed normal salt diet (NS; 0.49% NaCl) or HS (4% NaCl) for 2 weeks. NO signaling was assessed by measuring L‐NAME induced vasoconstriction of the afferent arteriole using the blood perfused juxtamedullary nephron (JMN) preparation. In this preparation, kidneys were perfused with blood from a donor rat on a matching or different diet to that of the kidney donor. Kidney endothelial cells were isolated with magnetic activated cell sorting and HDAC1 activity was measured. Results We found HS‐induced impaired NO signaling in the afferent arteriole. This was restored by inhibition of HDAC1 with MS‐275. Consistent with these findings, HDAC1 activity was increased in kidney endothelial cells. We further found the loss of NO to be dependent upon the diet of the blood donor rather than the diet of the kidney donor and the plasma from HS‐fed rats to be sufficient to induce impaired NO signaling. This indicates the presence of a humoral factor we termed plasma‐derived endothelial dysfunction mediator (PDEM). Pretreatment with the antioxidants, PEG‐SOD and PEG‐catalase, as well as the NOS cofactor, tetrahydrobiopterin, restored NO signaling. Conclusion We conclude that HS activates endothelial HDAC1 through PDEM leading to decreased NO signaling. This study provides novel insights into the molecular mechanisms by which a HS decreases renal microvascular endothelial NO signaling.

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.

GTP-Cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for tetrahydrobiopterin biosynthesis, an essential co-factor required for enzymatic activity of endothelial nitric oxide synthase (eNOS). Recent studies have suggested that endothelium-targeted overexpression of GTPCH I has vasoprotective effects. Therefore, we hypothesized that in endothelium GTPCH I localization in caveolae protects eNOS against oxidative stress. Human umbilical vein endothelial cells (HUVECs) were incubated with recombinant adenovirus encoding the human GTPCH I gene (Ad-GTPCH I, 100 MOI) or control recombinant adenoviral vector (Ad-ΔE1) for 12 hours in serum-free media. Cells were then fed with growth medium for 48 hours prior to analysis. Using sucrose gradient ultracentrifugation we detected both protein expression and enzymatic activity of GTPCH I in caveolar microdomains of HUVECs. Treatment of Ad-ΔE1 transduced endothelial cells for 24 hours with LY83583 (1 μ M), a superoxide anion generator, significantly decreased enzymatic activity of GTPCH I in caveolae-enriched membranes (0.49±0.12 pmol neopterin/mg protein; P<0.05 vs. control: 0.92±0.07 pmol neopterin/mg). Furthermore, LY83583 translocated eNOS from caveolae to cytosol in Ad-ΔE1 treated endothelial cells (P<0.05; n=3). This effect was specific because there was no change in caveolin-1 expression and no decrease in cell viability. Treatment of HUVECs with Ad-GTPCH I significantly increased GTPCH I activity in caveolae-enriched membranes (1.37±0.22 pmol neopterin/mg; P<0.05; n=4). Interestingly, LY83583-induced dislocation of eNOS from caveolar membrane fraction was significantly prevented by overexpression of GTPCH I (P<0.05; n=3). Our results suggest that overexpression of GTPCH I increases its enzymatic activity in caveolae. This effect preserves strategically key localization of eNOS in cell membrane during oxidative stress. This research has received full or partial funding support from the American Heart Association, National Center.

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.

Background: Optimal availability of tetrahydrobiopterin (BH4) is a critical determinant of nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) in the vascular endothelium. Biosynthesis of BH4 is regulated by the enzymatic activation of GTP cyclohydrolase I (GTPCH-I). While the physiological role of GTPCH-I and BH4 have been extensively characterized in peripheral vasculature, their role in regulation of cerebral vascular function has not been investigated. Methods: The role of GTPCH-I in regulation of cerebral vascular function was studied in cerebral microvessels isolated from wild-type (WT) mice and from mice with endothelium-targeted overexpression of GTPCH-I (eGTPCH-I Tg) mice. Vascular protein expression, intracellular levels of biopterin and cGMP (second messenger of NO) as well as production of NO and superoxide anions were determined. Results: Endothelium targeted over-expression of GTPCH-I resulted in significant increase in levels of BH4, as well as its oxidized product, 7,8-dihydrobiopterin (7,8-BH2). Importantly, ratio of BH4 to 7,8-BH2, indicative of BH4 available for eNOS activation, was significantly increased in cerebral microvessels of eGTPCH-I Tg mice. However, protein expression of eNOS, levels of nitrate/nitrite - indicative of NO production remained unchanged between cerebral microvessels of WT mice and eGTPCH-I Tg mice. Furthermore, increased BH4 biosynthesis did not affect production of superoxide anions or expression of antioxidant enzymes. Moreover, intracellular levels of cGMP, reflective of NO signaling and activation of soluble guanylate cyclase, were not affected in eGTPCH-I Tg mice. Conclusion: Our results suggest that, despite a significant increase in BH4 bioavailability, generation of endothelial NO in cerebral microvessels remained unchanged in eGTPCH-I Tg mice. We conclude that under physiological conditions the levels of BH4 are optimal for activation of eNOS and NO/cGMP signaling in wild-type mice.

Background: While reported neuroprotective effects of erythropoietin (EPO) make it an appealing candidate for its evaluation in protection of neurovascular unit during cerebrovascular or neurodegenerative disorders, effects of EPO on cerebral microvessels, the vascular component of neurovascular unit, have not been studied to date. The present study was designed to determine the effects of EPO in cerebral microvessels derived from wild-type mice and also from hph-1 mice, a genetic mouse model of BH4 deficiency. Methods: Hph-1 mice and wild-type littermates (C57BL/6 background) were administered recombinant human EPO (1000 U/kg/day) intraperitoneally for 3 days. Following treatment, mice were killed by injection of an overdose of pentobarbital, brains were removed and cerebral microvessels were isolated. Results: Treatment of wild-type mice with EPO did not affect BH4 bioavailability, superoxide anion production or basal cGMP levels. We have reported that cerebral microvessels of hph-1 mice demonstrated reduced bioavailability of BH4, increased production of superoxide anions and impaired endothelial NO/cGMP signaling. Treatment of hph-1 mice with EPO attenuated the levels of 7,8-dihydrobiopterin (7,8-BH2; P<0.05, n=5), oxidized product of BH4, and significantly increased the ratio of BH4 to 7,8-BH2 (P<0.05, n=5), indicative of increased bioavailability of BH4 for eNOS activation. Increased superoxide anion production in cerebral microvessels of hph-1 mice were attenuated by EPO treatment (P<0.05, n=5). While eNOS expression remained unchanged, levels of cGMP were significantly increased on EPO treatment in hph-1 mice (P< 0.05, n=6). Furthermore, EPO treatment selectively increased expression of manganese superoxide dismutase. Conclusion: The ability of EPO to attenuate oxidative stress and restore bioavailability of endothelial NO in cerebral microvessels may help to explain mechanisms responsible for cerebrovascular protective effects of EPO.