Katušić, Zvonimir S.

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.

Key Points The rat arteriovenous fistula (AVF) model exhibits marked upregulation of p16 Ink4a and p21 Cip1 and multiple markers of senescence. Fisetin, an established vasoprotective senolytic agent, when administered for 3 weeks, increases AVF blood flow and AVF outward remodeling. Heme is shown to be a novel prosenescence metabolite, and when chronically administered, it decreases AVF blood flow. Background Maturational failure of dialysis arteriovenous fistulas (AVFs) not uncommonly occurs and is of considerable and timely importance. Our prior studies demonstrate that senescence, a phenotypic process that promotes vascular and other diseases, occurs in the murine AVF. In this study, we examined whether senescence also occurs in the rat AVF model and the effect of compounds that inhibit or accelerate senescence. Methods The rat AVF was created in the femoral vessels by an end vein-side artery anastomosis. In the AVF, we assessed the expression of critical drivers of senescence, specifically, the cell cycle inhibitors p16 Ink4a and p21 Cip1 , and such indices of a senescence phenotype as senescence-associated β -galactosidase (SA- β -gal) activity, SA- β -gal staining, and a senescence-associated secretory phenotype. We examined the effects of compounds that retard or accelerate senescence on AVF blood flow. Results The AVF evinced upregulation of p16 Ink4a and p21 Cip1 when assessed 3 days after AVF creation. The AVF also demonstrated increased SA- β -gal activity in the artery and vein; staining for SA- β -gal in the AVF artery, anastomosis, and vein; and a prominent senescence-associated secretory phenotype. Fisetin, an established senolytic that is protective in other models of vascular injury, when administered for 3 weeks, increased AVF blood flow and outward remodeling. Hemin, when administered for 3 weeks, decreased AVF blood flow. We demonstrate that hemin is a novel inducer of a senescence phenotype in endothelial cells, as reflected by several senescence indices. However, when administered relatively acutely (for 5 days), hemin increased AVF blood flow by heme oxygenase–dependent mechanisms because the latter was entirely prevented by a competitive inhibitor of heme oxygenase activity. Conclusions The rat AVF exhibits senescence within 3 days of its creation. Chronic administration of a senolytic compound (fisetin) increases AVF blood flow, whereas chronic administration of a prosenescence compound (hemin) decreases AVF blood flow.

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.

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.

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.