Endothelium-independent Dilation Research Articles

Advanced age and reduced hyaluronan synthesis impair glycocalyx integrity resulting in endothelial dysfunction

The glycocalyx (GC) is a densely structured, gel-like matrix that coats the inner lining of the vascular endothelium. The GC plays a pivotal role in regulating the integrity of the vascular endothelium, primarily through mechano-sensing and -transducing shear stress, while also preserving endothelial barrier function. With advancing age, the GC deteriorates concomitantly with its essential constituent, hyaluronan (HA), which may lead to endothelial dysfunction. Therefore, we hypothesized that advanced age and reduced HA synthesis alone or together will impair GC integrity resulting in endothelial dysfunction. We have generated a tamoxifen-inducible, endothelial cell-specific, knockout mouse model of a hyaluronan producing enzyme, hyaluronan synthase 2 (HAS2), in young (4-6 mo) and old (22-23 mo, n=9-12/group). HAS2 mRNA expression in carotid endothelial cell lysates were reduced by ~47% in endothelial cell HAS2 knockout (ecHAS2KO) vs wildtype (ecHAS2WT, p=0.004) after tamoxifen treatment. The perfused boundary region (PBR), indicative of GC barrier function, was evaluated in vivo in the mesenteric microcirculation. The PBR was significantly impaired with aging (young vs old ecHAS2WT, p=0.047) and genotype (young ecHAS2WT vs KO, p=0.012) but there was no additive effect of aging and genotype (old ecHAS2WT vs KO, p=0.989). Subsequently, GC thickness was determined by measuring the depth of GC collapsed by leukocyte passage through the mesenteric segments. GC thickness was diminished by ~53% with aging (young vs old ecHAS2WT, p<0.0001) and ~59% with genotype (young ecHAS2WT vs KO, p<0.0001) but there was no synergistic effect of aging and genotype (old ecHAS2WT vs KO, p=0.893). Mechano-sensing and -transducing properties of the GC were assessed using ex vivo flow-induced vasodilation in isolated carotid arteries with and without the intra-luminal infusion of HA degrading enzyme, hyaluronidase (HYAL). Flow-induced vasodilation was diminished with aging (young vs old ecHAS2WT, p<0.0001) but this age-related decline was dampened with HYAL (p=0.212). The vasodilatory response to flow was reduced with genotype (young ecHAS2WT vs KO, p<0.0001) but this effect was weakened with HYAL (p=0.953). There was no augmentative effect of aging and genotype with and without HYAL (all p≥0.883). Acetylcholine induced endothelium-dependent dilation (EDD), a measure of endothelial function, was examined ex vivo in isolated mesenteric arteries. The maximal EDD was diminished by ~23% with aging (young vs old ecHAS2WT, p=0.04) but not genotype (young ecHAS2WT vs KO, p=0.962). There was an additive reduction in maximal EDD by ~46% with aging and genotype (old ecHAS2KO vs WT, p=0.021). Endothelium-independent dilation in response to sodium nitroprusside, an exogenous nitric oxide donor, was not different across groups with either aging, genotype, or both (ALL p>0.05). In conclusion, aging and reduced HA synthesis independently impaired GC integrity. As a result, mechano-sensing and -transducing property of GC was disturbed and further diminished by HYAL activity. In addition, endothelial function was impaired with aging and further exacerbated after a reduction in HA synthesis. Funded in part by awards from National Institutes of Health Awards R01 AG060395, R01 AG077751, R01 AG076748, T32 HL007576, T32 HL139451, Veteran's Affairs Merit Review Award I01 BX004492 and Nora Eccles Treadwell Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Circulating Milieu Mediates Arterial Dysfunction with Aging: Protection by Habitual Aerobic Exercise

Age-related arterial dysfunction (aortic stiffening and endothelial dysfunction) is the key antecedent to cardiovascular diseases. Habitual aerobic exercise largely mitigates arterial dysfunction in mid-life/older (ML/O) adults; however, the mechanisms by which this occurs remain to be fully elucidated. The circulating milieu (bioactive factors in the bloodstream) shifts towards a pro-inflammatory/oxidative state with advancing age but is preserved with habitual aerobic exercise. However, it is unclear if the circulating milieu directly contributes to age-related arterial dysfunction and if habitual aerobic exercise preserves arterial function with aging, in part, by favorably modulating the circulating milieu. PURPOSE: To determine the influence of aging and habitual aerobic exercise on circulating milieu-mediated aortic stiffening and endothelial function. METHODS: Human serum was collected from healthy young (18-29 yr) and ML/O (50+ yr) men and women. Aerobic exercise status was determined by leisure-time physical activity (>60 h/wk). Aortic rings (~1 mm) and common carotid arteries were obtained from young adult (3-4 mo), intervention naïve male and female C57BL/6N mice to assess aortic stiffness and endothelial function, respectively. Arteries were exposed ex vivofor 24-48 hours to 5% sex-matched human serum from one of the following groups: young sedentary (n=9; 4F), ML/O sedentary (n=14; 7F) or ML/O habitually aerobic exercise trained (n=8; 4F). Aortic stiffness was measured as elastic modulus (a measure of intrinsic mechanical wall stiffness). Endothelial function was assessed as endothelium-dependent dilation (EDD) and endothelium-independent dilation (EID) as peak response to acetylcholine and the nitric oxide (NO) donor sodium nitroprusside, respectively. Differences were determined via one-way ANOVA. RESULTS: Aortic Stiffness. Aortic elastic modulus was higher in aorta rings incubated with ML/O sedentary serum compared to young sedentary serum (ML/O sedentary, 4912 ± 588 v. Young sedentary, 3019 ± 522 kPa; p=0.020). Compared with ML/O sedentary serum, aorta rings incubated with ML/O trained serum had lower elastic modulus (2237 ± 426 kPa, p=0.002) which was not different to aorta rings incubated with young sedentary serum (p=0.369). Endothelial Function. Peak EDD was lower in carotid arteries incubated with ML/O sedentary v. young sedentary serum (79 ± 3% v. 92 ± 2%, p=0.001). Compared with ML/O sedentary serum, incubation with ML/O trained serum evoked higher peak EDD (93 ± 1%; p=0.001) that was similar to that produced by Young sedentary serum (p=0.866). There were no group differences in peak EID (p=0.228) suggesting that circulating milieu-induced differences were endothelium-specific and were not attributable to changes in smooth muscle sensitivity to NO. CONCLUSION: These findings suggest that the circulating milieu is a key mechanism by which aging induces arterial dysfunction and by which habitual aerobic exercise training preserves arterial function with aging. F31 HL165885, R21 AG078408, K99 HL159241, K99/R00 HL151818. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Age and APOE4 genotype alter cerebrovascular function and stiffness

Two of the greatest risk factors for late-onset Alzheimer’s disease (LOAD) are age and the APOE4 genotype. The gene encoding for apolipoprotein E ( APOE) has three isoforms in humans ( E2, E3, E4). E3 has the highest prevalence, while E4 exponentially increases the risk for AD. Additionally, it is well known that the E4 genotype is accompanied by an altered cerebral blood flow. However, the mechanisms underlying these blood flow changes in E4 individuals remain unclear and may be mediated by changes to cerebral artery structure and function. We aimed to determine if APOE genotype alters the impact of age on cerebral artery endothelial function and stiffness. We studied male and female, young E3 (n=20; ~6 months), aged E3 (n=9; ~24 months young E4 (n=22; ~6 months), and aged E4 (n=18; ~24 months) mice. We assessed endothelium-dependent and -independent vasodilation and vasoconstriction in isolated, pressurized posterior cerebral arteries (PCAs). Passive stiffness was measured in isolated PCAs after incubation in a calcium-free solution. Data are mean ± SD. Both old E3 and old E4 mice had impaired PCA endothelium-dependent vasodilation to acetylcholine compared with young mice ( E3: 29±10.6% vs 46±15.2%, p=0.02; E4: 30±10.8 vs 49±14.3%, p=0.0004). Sex comparisons found that the PCA maximal dilation to acetylcholine was 21% greater in young E3 females compared with young E3 males (52±12.5% vs 41±16%, p=0.04), and 13% greater in young E4 females compared with young E4 males (52±10% vs 45±16%, p=0.07), but there were no sex differences among the old groups. Additionally, the PCA responses to acetylcholine in the presence of nitric oxide synthase inhibitor L-NAME did not differ between groups (p>0.05). Endothelium-independent dilation, measured as the PCA response to sodium nitroprusside, also did not differ between groups (p>0.05). Aged E3 mice, compared with young E3, had a lower maximal constriction to potassium chloride (20±11% vs 59±16%, p<0.0001) and endothelin-1 (23±16% vs 53±14%, p=0.0005). In contrast, young E4 and old E4 mice had a similar maximal constriction to potassium chloride (35±13% vs 43±18%, p>0.05) and endothelin-1 (48±12% vs 47±20%, p>0.05). Lastly, we found that old mice had a greater PCA β stiffness index compared with young mice for both the E3 (15.5±4.5 vs 10.6±1.4 AU, p=0.0008) and E4 (13.4±2.9 vs 10.2±2.5 AU, p>0.0001) groups. In summary, we found that age impairs cerebral artery endothelial function in both APOE3 and APOE4 mice, and young female mice have better endothelial function than their male counterparts regardless of genotype. In addition, vasoconstrictor responsiveness appears to decline in old age in APOE3 mice, whereas vasoconstrictor responsiveness is maintained in old APOE4 mice. This maintained sensitivity to vasoconstrictors in old age may explain why APOE4 individuals have reduced cerebral blood flow and greater LOAD risk. Luvaas Family Fund, Alzheimer’s Association ALZDISCOVERY-1049110. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 122: Cerebral Artery Function And Stiffness Is Dependent On Apoe Genotype And Ovariectomy Status In Mice

The major risk factors for late-onset Alzheimer’s Disease (LOAD) include old age, female sex and the APOE4 allele. Post-menopausal females with an APOE4 allele have higher rates of LOAD compared with age-matched males with an APOE4 allele, suggesting an interaction between APOE genotype and estrogen deficiency. Importantly, cerebrovascular dysfunction is a contributor to LOAD risk. However, it is unknown if APOE genotype and estrogen deficiency interact to impact cerebrovascular function.Young female homozygous APOE3 and APOE4 mice (n=4-6 per group; ~6 months old) fed a high-fat, high-cholesterol diet were intact (sham) or ovariectomized (OVX). We assessed endothelium-dependent dilation to increasing doses of acetylcholine (ACh) in pressurized posterior cerebral arteries (PCAs). There was an interaction effect of APOE genotype and OVX status for endothelium-dependent dilation (p=0.03), such that APOE3 -Sham mice had a greater endothelium-dependent dilation compared to the APOE3 -OVX group (70±9.1% vs. 39±7.6%, p=0.003). These differences were mediated by nitric oxide as indicated by the absence of dilation in the presence of N omega-Nitro-L-arginine methyl ester hydrocholoride (L-NAME, Sham: 3.7±3.3% vs OVX: 4.1±4.0%, p= 0.99). In contrast, APOE4 -OVX had preserved endothelium-dependent dilation compared with APOE4 -Sham (49.9±14.8% vs 56.1±9.2%, p=0.8). Endothelium-independent dilation was assessed by the response to increasing doses of sodium nitroprusside and did not differ among groups (all p>0.05). Passive stiffness was measured in isolated PCAs after incubation in a calcium-free solution. For the elastic modulus at low pressures, there was an interaction effect of APOE genotype and OVX status (p=0.03), with APOE4 -OVX having a higher elastic modulus compared to APOE4 -Sham (0.93±0.3 vs. 0.47±0.1, p=0.03). The elastic modulus was similar in APOE3 groups (sham: 0.64±0.01, OVX: 0.62±0.09, p=0.99). Overall, we found that estrogen deficiency impacts cerebral artery endothelial function in APOE3 , but not APO E4 mice, while estrogen deficiency impacts cerebral artery stiffness in APOE4 , but not APOE3 mice. These results shed light on the impact of APOE genotype on the cerebral vasculature, suggesting a potential mechanism for LOAD.

Preserved endothelium-independent vascular function with aging in men and women: evidence from the peripheral and cerebral vasculature.

In the peripheral and cerebral vasculature, the impact of aging and sex on the endothelial-independent functional capacity of vascular smooth muscle cells (VSMCs) is not well understood, nor is it known whether such VSMC functions in these vascular beds reflect one another. Therefore, endothelium-independent dilation, at both the conduit (Δ diameter) and microvascular (Δ vascular conductance, VC) level, elicited by sublingual nitroglycerin (NTG, 0.8 mg of Nitrostat), compared with sham-delivery (control), was assessed using Doppler ultrasound in the popliteal (PA) and middle cerebral (MCA) artery of 20 young [23 ± 4 yr, 10 males (YM)/10 females (YF)] and 21 old [69 ± 5 yr, 11 males (OM)/10 females (OF)] relatively healthy adults. In the PA, compared with zero, NTG significantly increased diameter in all groups (YM: 0.29 ± 0.13, YF: 0.35 ± 0.26, OM: 0.30 ± 0.18, OF: 0.31 ± 0.14 mm), while control did not. The increase in VC only achieved significance in the OF (0.22 ± 0.31 mL/min/mmHg). In the MCA, compared with zero, NTG significantly increased diameter and VC in all groups (YM: 0.89 ± 0.30, 1.06 ± 1.28; YF: 0.97 ± 0.31, 1.84 ± 1.07; OM: 0.90 ± 0.42, 0.72 ± 0.99; OF: 0.74 ± 0.32, 1.19 ± 1.18, mm and mL/min/mmHg, respectively), while control did not. There were no age or sex differences or age-by-sex interactions for both the NTG-induced PA and MCA dilation and VC. In addition, PA and MCA dilation and VC responses to NTG were not related when grouped by age, sex, or as all subjects (r = 0.04-0.44, P > 0.05). Thus, peripheral and cerebral endothelial-independent VSMC function appears to be unaffected by age or sex, and variations in such VSMC function in one of these vascular beds are not reflected in the other.NEW & NOTEWORTHY To confidently explain peripheral and cerebral vascular dysfunction, it is essential to have a clear understanding of the endothelial-independent function of VSMCs across age and sex. By assessing endothelium-independent dilation using sublingual nitroglycerin, endothelial-independent VSMC function in the periphery (popliteal artery), and in the cerebral circulation (middle cerebral artery), was not different due to age or sex. In addition, endothelial-independent VSMC function in one of these vascular beds is not reflected in the other.

Cerebrovascular function is protected in hepatic xanthine oxidase knockout mice under chronic stress

Americans have been facing a mental health crisis that is predicted to impact and yield long-term psychological and physiological consequences negatively. We have shown that exposure to chronic stress can induce a pro-oxidative environment leading to cerebrovascular dysfunction and cognitive impairment. Xanthine oxidoreductase (XOR) is an enzyme that generates xanthine oxidase (XO) and xanthine dehydrogenase (XDH). Under hypoxic or pro-inflammatory conditions, XO produces excessive amounts of hydrogen peroxide and superoxide, which plays an important role in this stress-induced cerebrovascular dysfunction. Pharmacologically inhibiting XO with Febuxostat (50 mg/L) prevented stress-induced cerebrovascular dysfunction. XO is found in multiple tissues/organs; however, the liver is the major source of XO. Thus, this study aimed to understand how XO produced from the liver impacted cerebrovascular function during chronic stress. We hypothesize that liver XO production mediates stress-induced cerebrovascular dysfunction. To test our hypothesis, we exposed wild-type (WT) and hepatic XO knockout mice (HXO) to eight weeks of unpredictable chronic mild stress (UCMS) with or without Febuxostat (Feb). Groups were separated by HXO-control, HXO-UCMS, HXO-UCMS+Feb, WT-con, WT-UCMS, and WT-UCMS+Feb. During terminal procedures, the middle cerebral artery (MCA) was isolated and assessed using pressure myography to test the endothelial-dependent (EDD) and endothelium-independent dilation (EID) to acetylcholine (ACh) and sodium nitroprusside (SNP). The isolated MCA was kept under physiological conditions (pH 7.4±0.2; 70±2 mmHg, 37±1℃). In WT mice, UCMS decreased by 50% EDD (p<0.0001) and did not affect EID compared to WT-con, whereas the WT-UCMS+Feb group was fully protected, with similar EDD and EID values compared to WT-con. In contrast, HXO mice were partially protected from UCMS, reflected by only a 24% decrease (p<0.0001) in EDD and no change in EID. However, the HXO-UCMS+Feb group was fully protected with similar EDD and EID values to control mice. Our data shows that the liver significantly contributes to stress-induced cerebrovascular dysfunction. However, additional sources of XO circulation within the cerebrovasculature need further investigation. NIH, NINDS BINP R01 NS1117754, Psychosocial Stress-Induced Vascular Contributions to Cognitive Impairment and Alzheimer's. Disease: The Role of Xanthine Oxidase, NIH F31 Diversity Award, US DHHS-NIH-National Institute on Aging, Role of Xanthine Oxidase on Vascular Contributions to Cognitive Impairment This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Attenuated hyaluronan synthesis impairs glycocalyx integrity and leads to arterial dysfunction

The glycocalyx (GC) is a semi-solid layer lining the endothelium that plays a key role in maintaining barrier function and mechanosensing shear forces. With advanced age and pathological conditions, the GC deteriorates, and this is accompanied by arterial dysfunction characterized by impaired endothelial-dependent dilation (EDD) and large elastic artery stiffening. Alterations in hyaluronan (HA), a primary component of the GC, may contribute to the development of a dysfunctional artery phenotype. Here, we sought to determine the role of HA in GC integrity and arterial function. We have generated a tamoxifen-inducible, endothelial cell-specific, knockout mouse model of the hyaluronan producing enzyme, hyaluronan synthase 2 (HAS2, HAS2 f/f- Vecad-CreERT2). Endothelial HAS2 knockout (HAS2 f/f- Vecad-creERT2+: ecHAS2 KO, n=10) and wildtype (HAS2 +/+- Vecad-creERT2+: ecHAS2 WT, n=10) young mice were injected intraperitoneally with 4 mg of tamoxifen for 5 consecutive days. HAS2 mRNA expression was reduced by ~47% in carotid endothelial cells of ecHAS2 KO mice (P=0.004). GC barrier function (perfused boundary region [PBR]) was evaluated in vivo in the mesenteric microcirculation using an intravital microscope equipped with an automated capture and analysis system. PBR was 8.9% higher in ecHAS2 KO compared to ecHAS2 WT (P<0.0001). Subsequently, video recordings acquired were used to manually determine GC thickness from individual microvascular segments. GC thickness was significantly reduced in ecHAS2 KO vs ecHAS2 WT (7.9μm±0.02 vs 0.37μm±0.02, P<0.0001). Together, these results indicated that GC integrity was deteriorated in response to a reduction in endothelial HAS2. This GC deterioration impairs EDD, as evidenced by a 43.4% reduction in flow-induced dilation in isolated carotid arteries from ecHAS2 KO compared to ecHAS2 WT (P=0.013). To directly assess the role of GC plays in the differences in flow-induced EDD observed, the lumen of the arteries was infused with hyaluronidase, a HA degrading enzyme, and this abolished the differences in EDD between groups (P>0.05). Endothelial-independent dilation in response to sodium nitroprusside was not different between groups (P>0.05). Furthermore, aortic pulse wave velocity, a measure of large elastic artery stiffness, as well as systolic, diastolic, and mean arterial blood pressure were all greater in ecHAS2 KO compared to ecHAS2 WT (all P≤0.033). These results demonstrate that endothelial cell-specific impairment in HA synthesis compromises GC integrity and leads to arterial dysfunction and elevated blood pressure. Funded in part by awards from National Institutes of Health Awards R01 AG060395, R01 AG048366, T32 HL007576, T32 HL139451 and Veteran's Affairs Merit Review Award I01 BX004492. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Outbred, UM-HET3 mice have greater metabolic and arterial function than inbred, C57BL/6J mice

In recent years, UM-HET3 mice have become more commonly used in biomedical research. UM-HET3 mice are an outbred, genetically diverse strain of mice that is produced by crossing four inbred strains: BALB/cByJ, C57BL/6J, C3H/HeJ, and DBA/2J. Notably, the UM-HET3 strain is used by the National Institute on Aging Interventions Testing Program to investigate treatments that may extend lifespan and delay disease/dysfunction, while minimizing the impact of strain-specific phenotypes that might be observed in inbred, genetically identical strains like the C57BL/6J strain of mice. Here, we sought to examine phenotypic differences between age-matched male and female inbred, C57BL/6J and outbred, UM-HET3 mice (age: 6.4±0.0 mo, n=16 [8M/8F] per group). UM-HET3 had a greater body mass (C57BL/6J: 27±1 vs. UM-HET3: 32±1 g, P<0.05) and tibial length (C57BL/6J: 18.1±0.2 vs. UM-HET3: 19.6±0.3 mm, P<0.05) than C57BL/6J mice. Despite larger body stature, UM-HET3 mice had lower fasting blood glucose (C57BL/6J: 192±6 vs. UM-HET3: 168±5 mg/dL, P<0.05) and lower glucose area under the curve (AUC) in response to glucose tolerance testing (C57BL/6J: 34±1 vs. UM-HET3: 28±2 AU, P<0.05). On the contrary, there were no strain-related differences in glucose AUC in response to insulin tolerance testing (C57BL/6J: 14±1 vs. UM-HET3: 14±1 AU, P>0.05). We also observed no differences in systolic blood pressure (C57BL/6J: 115±2 vs. UM-HET3: 114±2 mmHg, P>0.05), although aortic stiffness, determined by pulse wave velocity (PWV), was lower in UM-HET3 compared to C57BL/6J mice (C57BL/6J: 309±5 vs. UM-HET3: 255±4 cm/sec, P<0.05). Endothelium-dependent dilation (EDD), determined by ex vivo maximal carotid artery dilation to acetylcholine (ACh), was lower in C57BL/6J compared to UM-HET3 (C57BL/6J: 84±2 vs. UM-HET3: 90±1%, P<0.05). In the presence of the nitric oxide (NO) synthase inhibitor, L-NAME, maximal dilation to ACh was similar between groups (C57BL/6J: 38±4 vs. UM-HET3: 34±4%, P>0.05). Thus, NO-mediated dilation, determined by subtracting ACh+L-NAME maximal dilation from ACh maximal dilation, was higher in UM-HET3 compared to C57BL/6J mice (C57BL/6J: 45±3 vs. UM-HET3: 57±4%, P<0.05). We observed no differences in endothelium-independent dilation, determined by ex vivo maximal dilation to sodium nitroprusside (C57BL/6J: 87±1 vs. UM-HET3: 90±2%, P>0.05). In summary, C57BL/6J mice appear to be smaller in stature, but with worse glucose handling, as indicated by impaired fasting glucose and glucose tolerance, compared to UM-HET3 mice. There are also strain differences in arterial function, as aortic stiffness was higher and EDD was lower in C57BL/6J compared to UM-HET3 mice. Strain differences in EDD appear to be due to a reduction in NO-mediated dilation in C57BL/6J mice. Collectively, these data suggest that there are phenotypic differences between these strains, indicating worse metabolic and arterial function in inbred, C57BL/6J compared to outbred, UM-HET3 mice. This study was funded in part by a grant from the National Institutes of Health (R00 AT010017). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

An acute bout of prolonged sitting blunts popliteal endothelium-independent dilation in young, healthy adults.

A single bout of prolonged uninterrupted sitting increases oxidative stress, reduces popliteal blood flow-induced shear stress, and diminishes endothelium-dependent, flow-mediated dilation (FMD). The FMD response is also influenced by the sensitivity of vascular smooth muscle cells to nitric oxide (i.e., endothelium-independent dilation), which is also attenuated by elevated oxidative stress. However, it is currently unknown whether prolonged sitting impacts popliteal endothelium-independent dilation responses, which may uncover a novel mechanism associated with sitting-induced vascular dysfunction. This study tested the hypothesis that prolonged sitting attenuates both popliteal FMD and endothelium-independent, nitroglycerin-mediated dilation responses (NMD, 0.4 mg sublingual dose). Popliteal blood flow (mL/min), relative FMD (%), and NMD (%) were assessed via duplex ultrasonography before and after a ∼3-h bout of sitting in 14 young, healthy adults (8♀; 22 ± 2 yr). Prolonged sitting attenuated resting blood flow (57 ± 23 to 32 ± 16 mL/min, P < 0.001), relative FMD (4.6 ± 2.8% to 2.2 ± 2.5%; P = 0.001), and NMD (7.3 ± 4.0% to 4.6 ± 3.0%; P = 0.002). These novel findings demonstrate that both endothelium-dependent and independent mechanisms contribute to the adverse vascular consequences associated with prolonged bouts of sitting.NEW & NOTEWORTHY We demonstrate that lower-limb vascular smooth muscle function is attenuated in young, healthy adults after an acute bout of prolonged sitting. These data indicate that prolonged sitting-induced vascular dysfunction involves both endothelium-dependent and -independent mechanisms.

Preserved Endothelium-independent Function With Aging: Evidence From The Peripheral And Cerebral Vasculature

There is growing evidence that vascular endothelial dysfunction is not only antecedent to cardiovascular disease, but also cognitive dysfunction and Alzheimer’s disease. Vascular smooth muscle cells (VSMCs) translate the endothelial-derived signal to vasodilate into action. PURPOSE: However, in the peripheral, and especially the cerebral, vasculature, the impact of aging on the functional capacity of VSMCs, and any functional link between these vascular beds, is not well understood. METHODS: Therefore, endothelium-independent dilation, elicited by sublingual nitroglycerin (NTG, 2 tabs, 0.4 mg Nitrostat each), compared to sham-delivery (control), was assessed using Doppler ultrasound in both the popliteal and middle cerebral (MCA) arteries of 12 young (22 ± 4 yrs, 6 male/6 female) and 11 old (67 ± 3 yrs, 6 male/5 female) healthy adults. RESULTS: In the popliteal artery, when compared to control (∆ young: 0.00 ± 0.03 mm, ∆ old: 0.02 ± 0.05 mm), NTG resulted in significant increases in vessel diameter (∆ young: 0.32 ± 0.13 mm, ∆ old: 0.37 ± 0.13 mm) with no differences between age or sex. Similarly, in the MCA, when compared to control (∆ young: 0.07 ± 0.11 mm, ∆ old: 0.02 ± 0.20 mm), NTG resulted in significant increases in vessel diameter (∆ young: 1.01 ± 0.30 mm, ∆ old: 1.00 ± 0.38 mm) with no differences between age or sex. Interestingly, there was no evidence of a relationship between the peripheral and cerebral vascular responses to NTG in terms of either age, sex, or all subjects as a whole. CONCLUSION: Thus, the known age-related vascular dysfunction, in the peripheral and cerebral circulations, does not appear to be the consequence of dysfunctional VSMCs, and there is no evidence of a link between VSMC function in these two vascular beds.

Short-term effects of electronic cigarettes on cerebrovascular function: A time course study.

What is the central question of this study? Acute exposure to electronic cigarettes (Ecigs) triggers abnormal vascular responses in systemic arteries; however, effects on cerebral vessels are poorly understood and time for recovery is not known. We hypothesized that exposure to cigarettes or Ecigs would trigger rapid (<4h) impairment of the middle cerebral artery (MCA) but that this would resolve by 24h. What is the main finding and its importance? Cigarettes and Ecigs caused similar degree and duration of MCA impairment. We find it takes ~72hours after exposure for MCA function to return to normal. This suggests that Ecig use is likely to produce similar adverse vascular health outcomes to those seen with cigarette smoke. Temporal influences of electronic cigarettes (Ecigs) on blood vessels are poorly understood. In this study, we evaluated a single episode of cigarette versus Ecig exposure on middle cerebral artery (MCA) reactivity and determined how long after the exposure MCA responses took to return to normal. We hypothesized that cigarette and Ecig exposure would induce rapid (<4h) reduction in MCA endothelial function and would resolve within 24h. Sprague-Dawley rats (4months old) were exposed to either air (n=5), traditional cigarettes (20 puffs, n=16) or Ecigs (20-puff group, n=16; or 60-puff group, n=12). Thereafter, the cigarette and Ecig groups were randomly assigned for postexposure vessel myography testing on day0 (D0, 1-4h postexposure), day1 (D1, 24-28h postexposure), day2 (D2, 48-52h postexposure) and day3 (72-76h postexposure). The greatest effect on endothelium-dependent dilatation was observed within 24h of exposure (∼50% decline between D0 and D1) for both cigarette and Ecig groups, and impairment persisted with all groups for up to 3days. Changes in endothelium-independent dilatation responses were less severe (∼27%) and shorter lived (recovering by D2) compared with endothelium-dependent dilatation responses. Vasoconstriction in response to serotonin (5-HT) was similar to endothelium-independent dilatation, with greatest impairment (∼45% for all exposure groups) at D0-D1, returning to normal by D2. These data show that exposure to cigarettes and Ecigs triggers a similar level/duration of cerebrovascular dysfunction after a single exposure. The finding that Ecig (without nicotine) and cigarette (with nicotine) exposure produce the same effects suggesting that nicotine is not likely to be triggering MCA dysfunction, and that vaping (with/without nicotine) has potential to produce the same vascular harm and/or disease as smoking.

Fisetin Supplementation Improves Age‐Related Vascular Endothelial Function by Suppressing Cellular Senescence and Mitochondrial Oxidative Stress

Age‐related vascular endothelial dysfunction is mediated by excess reactive oxygen species (ROS)—mitochondria (mito) being a key source—which can reduce nitric oxide (NO) bioavailability. Cellular senescence, a fundamental mechanism of aging, may exacerbate mito ROS and be a potential therapeutic target to combat age‐related vascular dysfunction.PurposeTo determine if treatment with the natural flavonoid fisetin improves endothelial function with aging by suppressing cellular senescence, scavenging excess whole‐cell and mito ROS, and increasing NO bioavailability.Methods &amp; ResultsOld (27 mo) male C57BL/6 mice were treated with vehicle (V; 10% EtOH, 30% PEG400 and 60% Phosal 50 PG; n = 9) or fisetin (Fis; 50 mg/kg/day in V; n = 10) by oral gavage following a 1 wk on – 2 wk off – 1 wk on dosing paradigm.Endothelial functionEndothelial function was assessed by ex vivo carotid artery endothelium‐dependent dilation (EDD) and endothelium‐independent dilation (EID) to increasing doses of acetylcholine and sodium nitroprusside, respectively. EDD was greater in Fis vs V treated mice (Peak EDD [%]: 97 ± 1 vs 84 ± 3, P &lt; .05) and no differences were observed among groups in EID (P = .54).Cellular senescenceFis‐treated mice had lower vascular abundance of p16, an established marker of cellular senescence (Fis, .12 ± .01 vs V, .19 ± .02 chemiluminescence units [CU], P &lt; .05). Next, we administered Fis or V to old (27 mo) p16‐3MR mice (a model that allows for clearance of senescent cells with ganciclovir [GCV]; n = 6/group). Fis‐treated p16‐3MR mice had greater EDD (Peak EDD [%]: Fis, 93 ± 2 vs V, 74 ± 5, P &lt; .05). Ex vivo carotid artery incubations with GCV eliminated group differences in EDD, suggesting that Fis reduced senescent cells to improve EDD.Whole‐cell ROSElectron paramagnetic resonance (EPR) spectroscopy was used to assess whole‐cell vascular (aortic) ROS. Fis‐treated mice had lower whole‐cell ROS (Fis, 4703 ± 455 vs V, 8173 ± 1243 amplitude units [AU], P &lt; .05). Incubation with the ROS scavenger TEMPOL eliminated group differences in EDD, implying that Fis ameliorated ROS‐related suppression of EDD.Mito ROSEPR was used to assess vascular mito ROS. Fis‐treated mice had lower mito ROS (Fis, 2527 ±440 vs V, 6603 ± 1956 AU, P &lt; .05). Further, Fis‐treated mice had lower abundance of vascular p‐p66SHC, a recognized marker of mito oxidative stress (Fis, .029 ± .003 vs V, .049 ± .008 CU, P &lt; .05) and greater abundance of Mn superoxide dismutase, a mito antioxidant enzyme (Fis, .41 ± .1 vs V, .20 ± .02 CU, P &lt; .05). Incubation with the mito‐specific antioxidant, MitoQ, eliminated group differences in EDD, implying that Fis ameliorated mito ROS‐related suppression of EDD.NOAddition of the NO‐synthase inhibitor, L‐NAME, abolished group differences in EDD suggesting Fis improved age‐related EDD impairments by increasing NO bioavailability.ConclusionFisetin supplementation may be a novel strategy to target excess cellular senescence and thereby reduce mito ROS to improve NO‐mediated endothelial function with aging.

In vivo arterial stiffness, but not isolated artery endothelial function, varies with the mouse estrus cycle

Due to the increasing inclusion of female mice in research, it is important to understand the influence of the estrus cycle on vascular function. In humans, endothelial function and arterial stiffness vary throughout the menstrual cycle; however, these functional changes have yet to be investigated for the mouse estrus cycle. During the estrus cycle, circulating estrogen is highest during the proestrus and estrus phases and lowest during diestrus, while progesterone peaks during estrus. Estrogens bind to estrogen receptor α (ERα), estrogen receptor β (ERβ), and estrogen G‐protein coupled receptor (GPR30) in vascular cells. Activation of ERα and GPR30 increases nitric oxide (NO) bioavailability. The purpose of this study was to determine the effect of the estrus cycle phase on arterial stiffness, vascular endothelial function, and estrogen receptor expression.In young female C57Bl/6J mice (n=23; 5.96 ± 0.04 months), we determined the estrus cycle via vaginal cytology and plasma hormone concentrations. We assessed large artery stiffness via aortic pulse wave velocity (PWV). Endothelial function was assessed by dose responses to acetylcholine (ACh) and insulin in pressurized mesenteric arteries (MAs) and posterior cerebral arteries (PCAs). NO‐mediated dilation was determined by the difference in vasodilation to ACh in the presence vs. absence of the NO synthase inhibitor L‐NAME. Endothelium independent dilation was assessed by the dose responses to sodium nitroprusside. We quantified gene expression of ERα (Esr1), ERβ (Esr2), and GPR30 (Gper1) in MAs and PCAs.Plasma progesterone concentrations were higher for mice in estrus (5.7 ± 1.1 ng/mL) compared to proestrus (2.5 ± 0.6 ng/mL; p=0.04) and tended to be higher in diestrus (3.9 ± 1.1 ng/mL, p=0.14), verifying the cytology classifications. Plasma 17β‐estradiol and estrone were below detection in most mice. There was a higher plasma testosterone concentration during diestrus (0.08 ± 0.02 ng/mL) compared to other stages (estrus 0.02 ± 0.00 ng/mL, p=0.006; proestrus 0.01 ± 0.00 ng/mL, p=0.001).Aortic PWV was lower for mice in estrus compared with mice in diestrus (3.0 ± 0.1 vs. 3.4 ± 0.1 m/s, p=0.005). Aortic PWV for mice in proestrus did not differ from the other stages (3.2 ± 0.1 m/s, p&gt;0.05). The MA and PCA responses to ACh, insulin and sodium nitroprusside, as well as NO‐mediated dilation, were not different between estrus cycle phases (p&gt;0.05). PCA Esr2 gene expression for mice in estrus was 57% lower than diestrus (p=0.008) and 54% lower than proestrus (p=0.04). Gene expression did not differ between estrus cycle phases for Esr1 and Gper1 in either the MA or PCA (p&gt;0.05), and MA Esr2 was not detectable.In summary, we found that the estrus phase in mice is associated with lower in vivo large artery stiffness. In contrast, there were no differences between estrus cycle phase in ex vivo resistance artery endothelial function. Changes to estrogen receptor gene expression during the estrus cycle are limited to ERβ in the cerebral arteries. These results suggest that estrus phase should be considered when measuring in vivo arterial stiffness in young female mice.

Stratification by Race Reveals Disparate Vascular Toxicity in Response to Anti‐Cancer Therapies

IntroductionCardiovascular toxicity is the primary reason for halting anti‐cancer therapy (CTx) or limiting the dose, and cardiovascular side effects of CTx represent a major clinical problem with implications for survival and quality of life. Ongoing work in our laboratory suggests that CTx induces microvascular endothelial dysfunction, which is a well‐established predictor of future CV events. CV disease in general and CTx‐induced adverse events are significantly more prevalent in Black patients (B) relative to non‐Hispanic White patients (NHW). Despite clinical evidence of these health disparities, little to no data exist to explain the underlying causes. Therefore, we tested the novel hypothesis that CTx‐induced microvascular dysfunction is augmented in B, which may explain the increase in cardiotoxic events in this population compared to NHW.MethodsFresh human tissues were obtained as de‐identified surgical discard specimens. Peripheral adipose arterioles (100–200 µm) from healthy B and NHW were incubated ex vivowith the CTx agents, doxorubicin (Dox), trastuzumab (TZM), or paclitaxel (PTX) for 16–20 h and used for isolated arteriole preparations. Endothelium‐dependent vasodilation in response to flow (Flow‐Mediated Dilation, FMD; 5–100 cmH2O pressure gradient) or acetylcholine (ACh; 10−9 to 10−5 M) and smooth muscle‐dependent dilation to papaverine (100 µM) were evaluated. Data are presented as % max. diameter ± SEM, N. Statistical significance was established with two‐way ANOVA RM.ResultsUnder control conditions, there was no difference in microvascular FMD in healthy B (ACh: 89.8±4.0%, n=5; 85.8±1.8%, n=6) vs. NHW (ACh: 86.2±3.2%, n=6; 78.7±5.9%, n=8). Ex vivo exposure to Dox impaired endothelial function in arterioles from both B and NHW (ACh: 45.0±10.5%, n=4 and 34.2±9.8%, n=7; FMD: Figure 1A). TZM impaired endothelial function to a greater extent in arterioles from B (ACh: 36.1±7.1%, n=3; FMD: Figure 1B) compared to NHW (ACh: 44.4±8.6%, n=8; FMD: Figure 1B). Interestingly, PTX did not affect endothelial function in NHW (ACh: 91.5±1.6%, n=4; FMD: Figure 1C), but impaired endothelial function in arterioles from B (ACh: 54.7±12.9%, n=3; FMD: Figure 1C). Max dilator capacity to the endothelium‐independent dilator papaverine was not changed in either group (not shown).ConclusionOur findings reveal disparate effects of CTx agents on microvascular endothelial function from B and NHW patients. This raises the possibility that microvascular dysfunction may contribute to the known disparities in adverse CV clinical outcomes of cancer patients and potentially other CV pathologies (e.g., disparities in adverse events associated with PTX‐eluting stents.) Future work should differentiate between biological/genetic vs. social/environmental contribution to observed microvascular responses to CTx.

The effect of camelina oil on vascular function in essential hypertensive patients with metabolic syndrome: a randomized, placebo-controlled, double-blind study

The effects of a dietary supplementation with the vegetable ω-3 α-linolenic acid (ALA) on cardiovascular homeostasis are unclear. In this context, it would be interesting to assess the effects of camelina oil. This study aimed to assess the cardiovascular and metabolic effects of camelina oil in hypertensive patients with metabolic syndrome. In a double-blind, placebo-controlled randomized study, treated essential hypertensive patients with metabolic syndrome received, during 6 mo, either cyclodextrin-complexed camelina oil containing≈1.5g ALA/d (n=40) or an isocaloric placebo (n=41), consisting of the same quantity of cyclodextrins and wheat starch. Anthropometric data, plasma lipids, glycemia, insulinemia, creatininemia, TBARs, high-sensitivity C-reactive protein, and n-3, n-6, and n-9 fatty acids in erythrocyte membranes were measured. Peripheral and central blood pressures, arterial stiffness, carotid intima-media thickness, and brachial artery endothelium-dependent flow-mediated dilatation (FMD) and endothelium-independent dilatation were assessed. Compared with placebo, camelina oil increased ALA (mean±SD: 0±0.04 compared with 0.08±0.06%, P<0.001), its elongation product EPA (0±0.5 compared with 0.16±0.65%, P<0.05), and the n-9 gondoic acid (GA; 0±0.04 compared with 0.08±0.04%, P<0.001). No between-group difference was observed for cardiovascular parameters. However, changes in FMD were associated with the magnitude of changes in EPA (r=0.26, P=0.03). Compared with placebo, camelina oil increased fasting glycemia (-0.2±0.6 compared with 0.3±0.5mmol/L, P<0.001) and HOMA-IR index (-0.8±2.5 compared with 0.5±0.9, P<0.01), without affecting plasma lipids, or inflammatory and oxidative stress markers. Changes in HOMA-IR index were correlated with the magnitude of changes in GA (r=0.32, P<0.01). Nutritional intake remained similar between groups. ALA supplementation with camelina oil did not improve vascular function but adversely affected glucose metabolism in hypertensive patients with metabolic syndrome. Whether this adverse effect on insulin sensitivity is related to GA enrichment, remains to be elucidated.

Impact of ALLopurInol on ENdothelial funCtion in diabEtic patients affected with coronary artery disease: ALLIENCE trial

Allopurinol, a xanthine oxidase inhibitor, was shown to be useful in improving endothelial dysfunction in diabetic patients independently from their coronary status. We sought to assess the impact of allopurinol on endothelial function in diabetic patients with coronary artery disease (CAD) reputed to have severe endothelial dysfunction. We performed a simple-blind randomized trial enrolling patients with type 2 diabetes and CAD who underwent percutaneous coronary intervention. Patients were randomly assigned to either conventional optimal medical therapy alone (control group) or optimal medical therapy associated with high dose of allopurinol: 300 mg daily the first month then 600 mg daily the second month (allopurinol group). Endothelial function was assessed by flow-mediated dilation (FMD). The primary endpoint was the changes in FMD (Δ FMD) at 2 months. The secondary endpoints were the changes in endothelium-independent brachial dilation, and quality of life (QoL) as assessed by the Seattle Angina Questionnaire. A total of 64 patients (mean age 58.5 ± 8.3 years, 84.1% males) were randomly assigned to either allopurinol group ( n = 32) and control group ( n = 32). At baseline, FMD was comparable between the two groups ( P = 0.961). At 2 months, no significant change was observed in FMD between allopurinol group and control group ( P = 0.709). Regarding QoL, patients in allopurinol group showed better improvement in angina frequency ( P = 0.020) and disease perception ( P < 0.001) than controls. Subgroups analysis revealed that allopurinol was associated with significant improvement in FMD only in patients with poorly controlled diabetes (HBA1c > 8%) ( P = 0.010) and those with acid uric serum level ≤ 287 μmol/L ( P = 0.010) ( Fig. 1 ). Although allopurinol improved QoL (particularly angina frequency) in CAD diabetic patients, it did not improve the endothelial function except in those with poorly controlled diabetes and low acid uric serum level.

The Role of Follicle-stimulating Hormone in Vascular Dysfunction Observed in Hematopoietic Cell Transplant Recipients.

Childhood cancer survivors who receive a hematopoietic cell transplantation (HCT) are at increased risk for follicle-stimulating hormone (FSH) abnormalities, which may have a substantial negative impact on vascular function. The purpose of this study was to examine the association of vascular function with FSH in HCT recipients, non-HCT recipients and healthy controls. The study included childhood cancer survivors who were HCT recipients (n=24) and non-HCT recipients (n=308), and a control group of healthy siblings (n=211) all between 9 and 18 years old. Vascular measures of carotid artery structure and function (compliance and distensibility), brachial artery flow-mediated dilation and endothelial-independent dilation were measured using ultrasound imaging. A fasting blood sample was collected to measure hormone levels. FSH was significantly higher in HCT recipients compared with non-HCT recipients and healthy controls (P<0.01). Carotid compliance and distensibility were significantly lower in HCT and non-HCT recipients compared with healthy controls (P<0.05). Higher FSH was associated with decreased carotid compliance (P<0.05). This study's results suggest that higher levels of FSH in HCT recipients may result in significant reductions in vascular function compared with non-HCT recipients and healthy controls. Therefore, gonadotropin endocrine dysfunction, particularly abnormal FSH levels, may be an underlying mechanism of vascular dysfunction.

ATP and acetylcholine interact to modulate vascular tone and α1-adrenergic vasoconstriction in humans.

The vascular endothelium senses and integrates numerous inputs to regulate vascular tone. Recent evidence reveals complex signal processing within the endothelium, yet little is known about how endothelium-dependent stimuli interact to regulate blood flow. We tested the hypothesis that combined stimulation of the endothelium with adenosine triphosphate (ATP) and acetylcholine (ACh) elicits greater vasodilation and attenuates α1-adrenergic vasoconstriction compared with combination of ATP or ACh with the endothelium-independent dilator sodium nitroprusside (SNP). We assessed forearm vascular conductance (FVC) in young adults (6 women, 7 men) during local intra-arterial infusion of ATP, ACh, or SNP alone and in the following combinations: ATP + ACh, SNP + ACh, and ATP + SNP, wherein the second dilator was coinfused after attaining steady state with the first dilator. By design, each dilator evoked a similar response when infused separately (ΔFVC, ATP: 48±4; ACh: 57±6; SNP: 53±6 mL·min-1·100mmHg-1; P ≥ 0.62). Combined infusion of the endothelium-dependent dilators evoked greater vasodilation than combination of either dilator with SNP (ΔFVC from first dilator, ATP + ACh: 45±9 vs. SNP + ACh: 18 ± 7 and ATP + SNP: 26 ± 4 mL·min-1·100 mmHg-1, P < 0.05). Phenylephrine was subsequently infused to evaluate α1-adrenergic vasoconstriction. Phenylephrine elicited less vasoconstriction during infusion of ATP or ACh versus SNP (ΔFVC, -25 ± 3 and -29 ± 4 vs. -48±3%; P < 0.05). The vasoconstrictor response to phenylephrine was further diminished during combined infusion of ATP + ACh (-13 ± 3%; P < 0.05 vs. ATP or ACh alone) and was less than that observed when either dilator was combined with SNP (SNP + ACh: -26±3%; ATP + SNP: -31 ± 4%; both P < 0.05 vs. ATP + ACh). We conclude that endothelium-dependent agonists interact to elicit vasodilation and limit α1-adrenergic vasoconstriction in humans.NEW & NOTEWORTHY The results of this study highlight the vascular endothelium as a critical site for integration of vasomotor signals in humans. To our knowledge, this is the first study to demonstrate that combined stimulation of the endothelium with ATP and ACh results in enhanced vasodilation compared with combination of either ATP or ACh with an endothelium-independent dilator. Furthermore, we show that ATP and ACh interact to modulate α1-adrenergic vasoconstriction in human skeletal muscle in vivo.

The cold receptor TRPM8 activation leads to attenuation of endothelium-dependent cerebral vascular functions during head cooling

Using the cranial window technique, we investigated acute effects of head cooling on cerebral vascular functions in newborn pigs. Head cooling lowered the rectal and extradural brain temperatures to 34.3 ± 0.6°C and 26.1 ± 0.6°C, respectively. During the 3-h hypothermia period, responses of pial arterioles to endothelium-dependent dilators bradykinin and glutamate were reduced, whereas the responses to hypercapnia and an endothelium-independent dilator sodium nitroprusside (SNP) remained intact. All vasodilator responses were restored after rewarming, suggesting that head cooling did not produce endothelial injury. We tested the hypothesis that the cold-sensitive TRPM8 channel is involved in attenuation of cerebrovascular functions. TRPM8 is immunodetected in cerebral vessels and in the brain parenchyma. During normothermia, the TRPM8 agonist icilin produced constriction of pial arterioles that was antagonized by the channel blocker AMTB. Icilin reduced dilation of pial arterioles to bradykinin and glutamate but not to hypercapnia and SNP, thus mimicking the effects of head cooling on vascular functions. AMTB counteracted the impairment of endothelium-dependent vasodilation caused by hypothermia or icilin. Overall, mild hypothermia produced by head cooling leads to acute reversible reduction of selected endothelium-dependent cerebral vasodilator functions via TRPM8 activation, whereas cerebral arteriolar smooth muscle functions are largely preserved.

Mechanisms for the Age‐Related Differences in Cerebral Artery Endothelial Function After Exposure to Elevated Pulse Pressure

Greater large artery stiffness is correlated with increased cerebrovascular dysfunction and damage. One possible mechanism for this correlation is increased pulse pressure in cerebral resistance arteries, a result of less dampening of pulse pressure by the large arteries, leading to reduced cerebral artery endothelial function. Old age is associated with increases in large artery stiffness and cerebrovascular impairment; however, the effect of age on the response to elevated pulse pressure in cerebral arteries is unclear. Thus, we hypothesized that cerebral resistance arteries exhibit an age-related decline in endothelial function following exposure to pulsatile pressure. In young (n=41, 5-10 mo.) and old (n=26, 25-30 mo.) male C57BL6 mice, we measured cerebrovascular function by the ex vivo endothelium-dependent dilation (EDD) to acetylcholine (ACh) in isolated posterior cerebral arteries following exposure to static pressure (50 mmHg), low pulse pressure (50-75 mmHg), or high pulse pressure (37.5-87.5 mmHg)(400bpm). We also determined the effects of nitric oxide synthase inhibition (L-NAME) and decomposition of hydrogen peroxide (PEG-catalase) on the response to ACh. We measured endothelium-independent dilation (EID) by the dose response to sodium nitroprusside. We also measured posterior cerebral artery wall thickness in young and old mice using histological analysis. Compared with young mice, cerebral arteries from old mice had a 37.3% lower maximal EDD during exposure to static pressure (p<0.001), but EID did not differ with age (p=0.86). Exposure to low pulse pressure did not affect maximal EDD for either young or old cerebral arteries (p>0.05). In young cerebral arteries, maximal EDD was 43.9% lower after exposure to high pulse pressure compared with static pressure (p<0.005); however, high pulse pressure had no effect on maximal EDD in old cerebral arteries (p=0.39). Addition of L-NAME reduced maximal EDD in both old (p=0.001) and young (p<0.001) cerebral arteries during all pressure conditions, such that maximal EDD was no longer different between groups (p>0.05). In young cerebral arteries, the presence of PEG-catalase led to a lower maximal EDD in the static pressure (p=0.045) and low pulse pressure (p=0.001) conditions and trended towards greater maximal EDD in the high pulse pressure condition (p=0.08). Cerebral arteries from old mice had a 22% greater wall thickness compared with cerebral arteries from young mice (p=0.047). In conclusion, these results suggest high pulse pressure impairs endothelial function in cerebral resistance arteries from young mice, and this impairment results from lowered bioavailability of the products of nitric oxide synthase and reduced contribution by hydrogen peroxide to vasodilation. Contrary to our hypothesis, cerebral arteries from old mice were protected from the deleterious effects of high pulse pressure, perhaps as a result of arterial remodeling in response to chronic exposure to higher pulse pressure in vivo.