Amy L. Sindler

Adiponectin Does Not Cross the Blood-Brain Barrier but Modifies Cytokine Expression of Brain Endothelial Cells

Adiponectin has recently been reported to generate a negative energy balance by increasing energy expenditure. However, it is unclear whether such effects require the presence and direct action of the adiponectin protein in the central nervous system. In this study, neither radiolabeled nonglycosylated nor glycosylated globular adiponectin crossed the blood-brain barrier (BBB) in mice. In addition, adiponectin was not detectable in human cerebrospinal fluid using various established methods. Using murine cerebral microvessels, we demonstrated expression of adiponectin receptors, which are upregulated during fasting, in brain endothelium. Interestingly, treatment with adiponectin reduced secretion of the centrally active interleukin-6 from brain endothelial cells, a phenomenon that was paralleled by a similar trend of other proinflammatory cytokines. In summary, our data suggest that direct effects of endogenous adiponectin on central nervous system pathways are unlikely to exist. However, the identification of adiponectin receptors on brain endothelial cells and the finding of a modified secretion pattern of centrally active substances from BBB cells provides an alternate explanation as to how adiponectin may evoke effects on energy metabolism.

Nitrite supplementation reverses vascular endothelial dysfunction and large elastic artery stiffness with aging.

We tested the hypothesis that short‐term nitrite therapy reverses vascular endothelial dysfunction and large elastic artery stiffening with aging, and reduces arterial oxidative stress and inflammation. Nitrite concentrations were lower (P < 0.05) in arteries, heart, and plasma of old (26–28 month) male C57BL6 control mice, and 3 weeks of sodium nitrite (50 mg L−1 in drinking water) restored nitrite levels to or above young (4–6 month) controls. Isolated carotid arteries of old control mice had lower acetylcholine (ACh)‐induced endothelium‐dependent dilation (EDD) (71.7 ± 6.1% vs. 93.0 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (P < 0.05 vs. young), and sodium nitrite restored EDD (95.5 ± 1.6%) by increasing NO bioavailability. 4‐Hydroxy‐2,2,6,6‐tetramethylpiperidine 1‐oxyl (TEMPOL), a superoxide dismutase (SOD) mimetic, apocynin, a nicotinamide adenine dinucleotide phosphate‐oxidase (NADPH) inhibitor, and sepiapterin (exogenous tetrahydrobiopterin) each restored EDD to ACh in old control, but had no effect in old nitrite‐supplemented mice. Old control mice had increased aortic pulse wave velocity (478 ± 16 vs. 332 ± 12 AU, P < 0.05 vs. young), which nitrite supplementation lowered (384 ± 27 AU). Nitrotyrosine, superoxide production, and expression of NADPH oxidase were ∼100–300% greater and SOD activity was ∼50% lower in old control mice (all P < 0.05 vs. young), but were ameliorated by sodium nitrite treatment. Inflammatory cytokines were markedly increased in old control mice (P < 0.05), but reduced to levels of young controls with nitrite supplementation. Short‐term nitrite therapy reverses age‐associated vascular endothelial dysfunction, large elastic artery stiffness, oxidative stress, and inflammation. Sodium nitrite may be a novel therapy for treating arterial aging in humans.

Effects of ageing and exercise training on eNOS uncoupling in skeletal muscle resistance arterioles

Reduced availability of tetrahydrobiopterin (BH4) contributes to the age‐related decline of nitric oxide (NO)‐mediated vasodilatation of soleus muscle arterioles. Depending on availability of substrate and/or necessary co‐factors, endothelial nitric oxide synthase (eNOS) can generate NO and/or superoxide (O2−). We evaluated the effects of age and chronic exercise on flow‐induced vasodilatation and levels of NO and O2− in soleus muscle arterioles. Young (3 months) and old (22 months) male rats were exercise trained or remained sedentary (SED) for 10 weeks. Flow‐stimulated NO and O2−, as well as BH4 and l‐arginine content, were determined in soleus muscle arterioles. Flow‐induced vasodilatation was assessed under control conditions and during the blockade of O2− and/or hydrogen peroxide. Exercise training enhanced flow‐induced vasodilatation in arterioles from young and old rats. Old age reduced, and exercise training restored, BH4 content and flow‐stimulated NO availability. Flow‐stimulated, eNOS‐derived O2− levels were higher in arterioles from old SED compared to those from young SED rats. Exercise training increased flow‐stimulated eNOS‐derived O2− levels in arterioles from young but not old rats. O2− scavenging with Tempol reduced flow‐induced vasodilatation from all groups except young SED rats. Addition of catalase to Tempol‐treated arterioles eliminated flow‐induced vasodilatation in arterioles from all groups. Catalase reduced flow‐induced vasodilatation from all groups. In Tempol‐treated arterioles, flow‐induced vasodilatation was restored by deferoxamine, an iron chelator. These data indicate that uncoupling of eNOS contributes to the age‐related decline in flow‐induced vasodilatation; however, reactive oxygen species are required for flow‐induced vasodilatation in soleus muscle arterioles from young and old rats.

Exercise Protects Against Myocardial Ischemia–Reperfusion Injury via Stimulation of β3-Adrenergic Receptors and Increased Nitric Oxide Signaling: Role of Nitrite and Nitrosothiols

Rationale: Exercise training confers sustainable protection against ischemia–reperfusion injury in animal models and has been associated with improved survival following a heart attack in humans. It is still unclear how exercise protects the heart, but it is apparent that endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) play a role. Objective: To determine the role of &bgr;3-adrenergic receptors (&bgr;3-ARs), eNOS activation, and NO metabolites (nitrite and nitrosothiols) in the sustained cardioprotective effects of exercise. Methods and Results: Here we show that voluntary exercise reduces myocardial injury in mice following a 4-week training period and that these protective effects can be sustained for at least 1 week following the cessation of the training. The sustained cardioprotective effects of exercise are mediated by alterations in the phosphorylation status of eNOS (increase in serine 1177 and decrease in threonine 495), leading to an increase in NO generation and storage of NO metabolites (nitrite and nitrosothiols) in the heart. Further evidence revealed that the alterations in eNOS phosphorylation status and NO generation were mediated by &bgr;3-AR stimulation and that in response to exercise a deficiency of &bgr;3-ARs leads to an exacerbation of myocardial infarction following ischemia–reperfusion injury. Conclusions: Our findings clearly demonstrate that exercise protects the heart against myocardial ischemia–reperfusion injury by stimulation of &bgr;3-ARs and increased cardiac storage of nitric oxide metabolites (ie, nitrite and nitrosothiols).

Translational evidence that impaired autophagy contributes to arterial ageing

•  Advancing age is the major risk factor for the development of cardiovascular diseases. •  Arterial endothelial dysfunction, characterized by impaired endothelium‐dependent dilatation (EDD), is a key antecedent to age‐associated clinical cardiovascular disease. •  We tested the hypothesis that changes in autophagy, the process by which cells recycle damaged biomolecules, may be an underlying cause of the age‐related reduction in EDD. •  We show that autophagy is impaired in arteries of older humans and mice with reduced EDD, and that enhancing autophagy restores EDD by reducing superoxide‐dependent oxidative stress and inflammation, and increasing nitric oxide bioavailability. •  Our results identify impaired autophagy as a potential cause of age‐related arterial dysfunction and suggest that boosting autophagy may be a novel strategy for the treatment of arterial endothelial dysfunction and prevention of cardiovascular diseases with ageing.

Superoxide–lowering therapy with TEMPOL reverses arterial dysfunction with aging in mice

To test the hypothesis that the antioxidant enzyme superoxide dismutase (SOD) mimetic TEMPOL improves arterial aging, young (Y, 4–6 months) and old (O, 26–28 months) male C57BL6 mice received regular or TEMPOL‐supplemented (1mM) drinking water for 3 weeks (n = 8 per group). Aortic superoxide was 65% greater in O (P < 0.05 vs. Y), which was normalized by TEMPOL. O had large elastic artery stiffening, as indicated by greater aortic pulse wave velocity (aPWV, 508 ± 22 vs. 418 ± 22 AU), which was associated with increased adventitial collagen I expression (P < 0.05 vs. Y). TEMPOL reversed the age‐associated increases in aPWV (434 ± 21 AU) and collagen in vivo, and SOD reversed the increases in collagen I in adventitial fibroblasts from older rats in vitro. Isolated carotid arteries of O had impaired endothelial function as indicated by reduced acetylcholine‐stimulated endothelium‐dependent dilation (EDD) (75.6 ± 3.2 vs. 94.5 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (l‐NAME) associated with decreased endothelial NO synthase (eNOS) expression (P < 0.05 vs. Y). TEMPOL restored EDD (94.5 ± 1.4%), NO bioavailability and eNOS in O. Nitrotyrosine and expression of NADPH oxidase were ∼100–200% greater, and MnSOD was ∼75% lower in O (P < 0.05 vs. Y). TEMPOL normalized nitrotyrosine and NADPH oxidase in O, without affecting MnSOD. Aortic pro‐inflammatory cytokines were greater in O (P < 0.05 vs. Y) and normalized by TEMPOL. Short‐term treatment of excessive superoxide with TEMPOL ameliorates large elastic artery stiffening and endothelial dysfunction with aging, and this is associated with normalization of arterial collagen I, eNOS, oxidative stress, and inflammation.

Curcumin ameliorates arterial dysfunction and oxidative stress with aging

We tested the hypothesis that curcumin supplementation would reverse arterial dysfunction and vascular oxidative stress with aging. Young (Y, 4-6 months) and old (O, 26-28 months) male C57BL6/N mice were given normal or curcumin supplemented (0.2%) chow for 4 weeks (n=5-10/group/measure). Large elastic artery stiffness, assessed by aortic pulse wave velocity (aPWV), was greater in O (448±15 vs. 349±15 cm/s) and associated with greater collagen I and advanced glycation end-products and less elastin (all P<0.05). In O, curcumin restored aPWV (386±15 cm/s), collagen I and AGEs (AGEs) to levels not different vs. Y. Ex vivo carotid artery acetylcholine (ACh)-induced endothelial-dependent dilation (EDD, 79±3 vs. 94±2%), nitric oxide (NO) bioavailability and protein expression of endothelial NO synthase (eNOS) were lower in O (all P<0.05). In O, curcumin restored NO-mediated EDD (92±2%) to levels of Y. Acute ex vivo administration of the superoxide dismutase (SOD) mimetic TEMPOL normalized EDD in O control mice (93±3%), but had no effect in Y control or O curcumin treated animals. O had greater arterial nitrotyrosine abundance, superoxide production and NADPH oxidase p67 subunit expression, and lower manganese SOD (all P<0.05), all of which were reversed with curcumin. Curcumin had no effects on Y. Curcumin supplementation ameliorates age-associated large elastic artery stiffening, NO-mediated vascular endothelial dysfunction, oxidative stress and increases in collagen and AGEs in mice. Curcumin may be a novel therapy for treating arterial aging in humans.

Mitochondria-targeted antioxidant (MitoQ) ameliorates age-related arterial endothelial dysfunction in mice

The development of age‐related arterial endothelial dysfunction, a key antecedent of increased cardiovascular disease (CVD) risk, is mediated largely by reduced nitric oxide bioavailability as a consequence of oxidative stress. Mitochondria are critical signalling organelles in the vasculature, which, when dysregulated, become a source of excessive reactive oxygen species; the role of mitochondria‐derived oxidative stress in age‐related vascular dysfunction is unknown. We show that a mitochondria‐targeted antioxidant, MitoQ, ameliorates vascular endothelial dysfunction in old mice and that these improvements are associated with the normalization of mitochondria‐derived oxidative stress and markers of arterial mitochondrial health. These results indicate that mitochondria‐derived oxidative stress is an important mechanism underlying the development of age‐related vascular endothelial dysfunction and therefore may be a promising therapeutic target. Mitochondria‐targeted antioxidants represent a novel strategy for preserving healthy vascular endothelial function in primary ageing and preventing age‐related CVD in humans.

The SIRT1 activator SRT1720 reverses vascular endothelial dysfunction, excessive superoxide production, and inflammation with aging in mice

Reductions in arterial SIRT1 expression and activity with aging are linked to vascular endothelial dysfunction. We tested the hypothesis that the specific SIRT1 activator SRT1720 improves endothelial function [endothelium-dependent dilation (EDD)] in old mice. Young (4-9 mo) and old (29-32 mo) male B6D2F1 mice treated with SRT1720 (100 mg/kg body wt) or vehicle for 4 wk were studied with a group of young controls. Compared with the young controls, aortic SIRT1 expression and activity were reduced (P < 0.05) and EDD was impaired (83 ± 2 vs. 96 ± 1%; P < 0.01) in old vehicle-treated animals. SRT1720 normalized SIRT1 expression/activity in old mice and restored EDD (95 ± 1%) by enhancing cyclooxygenase (COX)-2-mediated dilation and protein expression in the absence of changes in nitric oxide bioavailability. Aortic superoxide production and expression of NADPH oxidase 4 (NOX4) were increased in old vehicle mice (P < 0.05), and ex vivo administration of the superoxide scavenger TEMPOL restored EDD in that group. SRT1720 normalized aortic superoxide production in old mice, without altering NOX4 and abolished the improvement in EDD with TEMPOL, while selectively increasing aortic antioxidant enzymes. Aortic nuclear factor-κB (NF-κB) activity and tumor necrosis factor-α (TNF-α) were increased in old vehicle mice (P < 0.05), whereas SRT1720 normalized NF-κB activation and reduced TNF-α in old animals. SIRT1 activation with SRT1720 ameliorates vascular endothelial dysfunction with aging in mice by enhancing COX-2 signaling and reducing oxidative stress and inflammation. Specific activation of SIRT1 is a promising therapeutic strategy for age-related endothelial dysfunction in humans.

Nicotinamide mononucleotide supplementation reverses vascular dysfunction and oxidative stress with aging in mice

We tested the hypothesis that supplementation of nicotinamide mononucleotide (NMN), a key NAD+ intermediate, increases arterial SIRT1 activity and reverses age‐associated arterial dysfunction and oxidative stress. Old control mice (OC) had impaired carotid artery endothelium‐dependent dilation (EDD) (60 ± 5% vs. 84 ± 2%), a measure of endothelial function, and nitric oxide (NO)‐mediated EDD (37 ± 4% vs. 66 ± 6%), compared with young mice (YC). This age‐associated impairment in EDD was restored in OC by the superoxide ( O2− ) scavenger TEMPOL (82 ± 7%). OC also had increased aortic pulse wave velocity (aPWV, 464 ± 31 cm s−1 vs. 337 ± 3 cm s−1) and elastic modulus (EM, 6407 ± 876 kPa vs. 3119 ± 471 kPa), measures of large elastic artery stiffness, compared with YC. OC had greater aortic O2− production (2.0 ± 0.1 vs. 1.0 ± 0.1 AU), nitrotyrosine abundance (a marker of oxidative stress), and collagen‐I, and reduced elastin and vascular SIRT1 activity, measured by the acetylation status of the p65 subunit of NFκB, compared with YC. Supplementation with NMN in old mice restored EDD (86 ± 2%) and NO‐mediated EDD (61 ± 5%), reduced aPWV (359 ± 14 cm s−1) and EM (3694 ± 315 kPa), normalized O2− production (0.9 ± 0.1 AU), decreased nitrotyrosine, reversed collagen‐I, increased elastin, and restored vascular SIRT1 activity. Acute NMN incubation in isolated aortas increased NAD+ threefold and manganese superoxide dismutase (MnSOD) by 50%. NMN supplementation may represent a novel therapy to restore SIRT1 activity and reverse age‐related arterial dysfunction by decreasing oxidative stress.