Interactions between zinc and NRF2 in vascular redox signalling.

Recent evidence highlights the importance of trace metal micronutrients such as zinc (Zn) in coronary and vascular diseases. Zn2+ plays a signalling role in modulating endothelial nitric oxide synthase and protects the endothelium against oxidative stress by up-regulation of glutathione synthesis. Excessive accumulation of Zn2+ in endothelial cells leads to apoptotic cell death resulting from dysregulation of glutathione and mitochondrial ATP synthesis, whereas zinc deficiency induces an inflammatory phenotype, associated with increased monocyte adhesion. Nuclear factor-E2-related factor 2 (NRF2) is a transcription factor known to target hundreds of different genes. Activation of NRF2 affects redox metabolism, autophagy, cell proliferation, remodelling of the extracellular matrix and wound healing. As a redox-inert metal ion, Zn has emerged as a biomarker in diagnosis and as a therapeutic approach for oxidative-related diseases due to its close link to NRF2 signalling. In non-vascular cell types, Zn has been shown to modify conformations of the NRF2 negative regulators Kelch-like ECH-associated Protein 1 (KEAP1) and glycogen synthase kinase 3ß (GSK3ß) and to promote degradation of BACH1, a transcriptional suppressor of select NRF2 genes. Zn can affect phosphorylation signalling, including mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinases and protein kinase C, which facilitate NRF2 phosphorylation and nuclear translocation. Notably, several NRF2-targeted proteins have been suggested to modify cellular Zn concentration via Zn exporters (ZnTs) and importers (ZIPs) and the Zn buffering protein metallothionein. This review summarises the cross-talk between reactive oxygen species, Zn and NRF2 in antioxidant responses of vascular cells against oxidative stress and hypoxia/reoxygenation.

Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels.

Unregulated increases in cellular Ca2+ homeostasis are a hallmark of pathophysiological conditions and a key trigger of cell death. Endothelial cells cultured under physiologic O2 conditions (5% O2) exhibit a reduced cytosolic Ca2+ response to stimulation. The mechanism for reduced plateau [Ca2+]i upon stimulation was due to increased sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)-mediated reuptake rather than changes in Ca2+ influx capacity. Agonist-stimulated phosphorylation of the SERCA regulatory protein phospholamban was increased in cells cultured under 5% O2. Elevation of cytosolic and mitochondrial [Ca2+] and cell death after prolonged ionomycin treatment, as a model of Ca2+ overload, were lower when cells were cultured long-term under 5% compared with 18% O2. This protection was abolished by cotreatment with the SERCA inhibitor cyclopiazonic acid. Taken together, these results demonstrate that culturing cells under hyperoxic conditions reduces their ability to efficiently regulate [Ca2+]i, resulting in greater sensitivity to cytotoxic stimuli.-Keeley, T. P., Siow, R. C. M., Jacob, R., Mann, G. E. Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels.

A Novel α-Calcitonin Gene-Related Peptide Analogue Protects Against End-Organ Damage in Experimental Hypertension, Cardiac Hypertrophy, and Heart Failure.

BACKGROUND: Research into the therapeutic potential of α-calcitonin gene-related peptide (α-CGRP) has been limited because of its peptide nature and short half-life. Here, we evaluate whether a novel potent and long-lasting (t½ ≥7 hours) acylated α-CGRP analogue (αAnalogue) could alleviate and reverse cardiovascular disease in 2 distinct murine models of hypertension and heart failure in vivo. METHODS: The ability of the αAnalogue to act selectively via the CGRP pathway was shown in skin by using a CGRP receptor antagonist. The effect of the αAnalogue on angiotensin II-induced hypertension was investigated over 14 days. Blood pressure was measured by radiotelemetry. The ability of the αAnalogue to modulate heart failure was studied in an abdominal aortic constriction model of murine cardiac hypertrophy and heart failure over 5 weeks. Extensive ex vivo analysis was performed via RNA analysis, Western blot, and histology. RESULTS: The angiotensin II-induced hypertension was attenuated by cotreatment with the αAnalogue (50 nmol·kg-1·d-1, SC, at a dose selected for lack of long-term hypotensive effects at baseline). The αAnalogue protected against vascular, renal, and cardiac dysfunction, characterized by reduced hypertrophy and biomarkers of fibrosis, remodeling, inflammation, and oxidative stress. In a separate study, the αAnalogue reversed angiotensin II-induced hypertension and associated vascular and cardiac damage. The αAnalogue was effective over 5 weeks in a murine model of cardiac hypertrophy and heart failure. It preserved heart function, assessed by echocardiography, while protecting against adverse cardiac remodeling and apoptosis. Moreover, treatment with the αAnalogue was well tolerated with neither signs of desensitization nor behavioral changes. CONCLUSIONS: These findings, in 2 distinct models, provide the first evidence for the therapeutic potential of a stabilized αAnalogue, by mediating (1) antihypertensive effects, (2) attenuating cardiac remodeling, and (3) increasing angiogenesis and cell survival to protect against and limit damage associated with the progression of cardiovascular diseases. This indicates the therapeutic potential of the CGRP pathway and the possibility that this injectable CGRP analogue may be effective in cardiac disease.

A PP2A-mediated feedback mechanism controls Ca2+-dependent NO synthesis under physiological oxygen.

Intracellular O2 is a key regulator of NO signaling, yet most in vitro studies are conducted in atmospheric O2 levels, hyperoxic with respect to the physiologic milieu. We investigated NO signaling in endothelial cells cultured in physiologic (5%) O2 and stimulated with histamine or shear stress. Culture of cells in 5% O2 (>5 d) decreased histamine- but not shear stress-stimulated endothelial (e)NOS activity. Unlike cells adapted to a hypoxic environment (1% O2), those cultured in 5% O2 still mobilized sufficient Ca2+ to activate AMPK. Enhanced expression and membrane targeting of PP2A-C was observed in 5% O2, resulting in greater interaction with eNOS in response to histamine. Moreover, increased dephosphorylation of eNOS in 5% O2 was Ca2+-sensitive and reversed by okadaic acid or PP2A-C siRNA. The present findings establish that Ca2+ mobilization stimulates both NO synthesis and PP2A-mediated eNOS dephosphorylation, thus constituting a novel negative feedback mechanism regulating eNOS activity not present in response to shear stress. This, coupled with enhanced NO bioavailability, underpins differences in NO signaling induced by inflammatory and physiologic stimuli that are apparent only in physiologic O2 levels. Furthermore, an explicit delineation between physiologic normoxia and genuine hypoxia is defined here, with implications for our understanding of pathophysiological hypoxia.-Keeley, T. P., Siow, R. C. M., Jacob, R., Mann, G. E. A PP2A-mediated feedback mechanism controls Ca2+-dependent NO synthesis under physiological oxygen.

Perturbations of the anti-ageing hormone Klotho in patients with type 1 diabetes and microalbuminuria.

AIMS/HYPOTHESIS: Patients with type 1 diabetes and microalbuminuria are at high risk of cardiovascular disease (CVD) and end-stage renal disease. Soluble Klotho is an anti-ageing circulating hormone involved in phosphate metabolism and vascular homeostasis through protective effects on the endothelium and antioxidant actions. The role of soluble Klotho in patients with type 1 diabetes and microalbuminuria is unknown. METHODS: In a cross-sectional single-centre study we evaluated the levels of circulating serum soluble Klotho in 33 participants with type 1 diabetes and a history of microalbuminuria (receiving renin-angiotensin system [RAS] inhibitors) and 45 participants with type 1 diabetes without a history of microalbuminuria (not receiving RAS or other antihypertensive drugs). All participants had an eGFR >45 ml/min, duration of diabetes >20 years and no history of CVD. Serum soluble Klotho levels were measured by a validated immunoassay. RESULTS: Participants with microalbuminuria had significantly lower levels of serum Klotho compared with those without microalbuminuria (median [interquartile range], 659.3 [525.3, 827.6] vs 787.7 [629.5, 1007]; p = 0.023). This difference persisted after adjustment for variables including age and eGFR. In a subgroup of 30 individuals with and without microalbuminuria, other markers of phosphate balance were not significantly different. CONCLUSIONS/INTERPRETATION: In individuals with type 1 diabetes, microalbuminuria is associated with soluble Klotho deficiency. Further studies are required to determine whether soluble Klotho is causally related to the development of cardio-renal disease in type 1 diabetes.

The anti-ageing hormone klotho induces Nrf2-mediated antioxidant defences in human aortic smooth muscle cells.

Vascular ageing in conditions such as atherosclerosis, diabetes and chronic kidney disease, is associated with the activation of the renin angiotensin system (RAS) and diminished expression of antioxidant defences mediated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). The anti-ageing hormone klotho promotes longevity and protects against cardiovascular and renal diseases. Klotho has been shown to activate Nrf2 and attenuate oxidative damage in neuronal cells, however, the mechanisms by which it protects against vascular smooth muscle cell VSMC dysfunction elicited by Angiotensin II (AngII) remain to be elucidated. AngII contributes to vascular ageing and atherogenesis by enhancing VSMC oxidative stress, senescence and apoptosis. This study demonstrates that soluble klotho (1 nM, 24 hrs) significantly induces expression of Nrf2 and the antioxidant enzymes haeme oxygenase (HO-1) and peroxiredoxin-1 (Prx-1) and enhances glutathione levels in human aortic smooth muscle cells (HASMC). Silencing of Nrf2 attenuated the induction of HO-1 and Prx-1 expression by soluble klotho. Furthermore, soluble klotho protected against AngII-mediated HASMC apoptosis and senescence via activation of Nrf2. Thus, our findings highlight a novel Nrf2-mediated mechanism underlying the protective actions of soluble klotho in HAMSC. Targeting klotho may thus represent a therapeutic strategy against VSMC dysfunction and cardiovascular ageing.

Deficiency of p62/Sequestosome 1 causes hyperphagia due to leptin resistance in the brain.

The cytoplasmic regulatory protein p62 (Sequestosome 1/A170) is known to modulate various receptor-mediated intracellular signaling pathways. p62 deficiency was shown to result in mature-onset obesity in mice, but the mechanisms underlying this abnormality remained unclear. Here we report that hyperphagia due to central leptin resistance is the cause of obesity in p62(-/-) mice. We found that these mice show hyperphagia. Restriction of food to the amount eaten by wild-type mice prevented excess body weight gain and fat accumulation, suggesting that overfeeding is the primary cause of obesity in p62(-/-) mice. Brain-specific p62 deficiency caused mature-onset obesity to the same extent as in p62(-/-) mice, further supporting a neuronal mechanism as the major cause of obesity in these mice. Immunohistochemical analysis revealed that p62 is highly expressed in hypothalamic neurons, including POMC neurons in the arcuate nucleus. Central leptin resistance was observed even in young preobese p62(-/-) mice. We found a defect in intracellular distribution of the transcription factor Stat3, which is essential for the action of leptin, in p62(-/-) mice. These results indicate that brain p62 plays an important role in bodyweight control by modulating the central leptin-signaling pathway and that lack of p62 in the brain causes leptin resistance, leading to hyperphagia. Thus, p62 could be a clinical target for treating obesity and metabolic syndrome.

The Longevity Med Summit: insights on healthspan from cell to society.

In recent years, there has been a paradigm shift with regards to ageing, challenging its traditional perception as an inevitable and natural process. Researchers have collectively identified hallmarks of ageing, nine of which were initially proposed in 2013 and expanded in 2023 to include disabled macroautophagy, chronic inflammation, and dysbiosis, enhancing our understanding of the ageing process at microscopic, cellular, and system-wide levels. Strategies to manipulate these hallmarks present opportunities for slowing, preventing, or reversing age-related diseases, thereby promoting longevity. The interdependence of these hallmarks underscores the necessity of a comprehensive, systems-based approach to address the complex processes contributing to ageing. As a primary risk factor for various diseases, ageing diminishes healthspan, leading to extended periods of compromised health and multiple age-related conditions towards the end of life. The significant gap between healthspan and lifespan holds substantial economic and societal implications. The inaugural Longevity Med Summit (4-5 May 2023, Cascais, Portugal) provided an international forum to discuss the academic and industry landscape of healthy longevity research, preventive medicine and clinical practice to enhance healthspan.

A Randomised, Double-Blind, Placebo-Controlled, Cross-Over Clinical Trial to Evaluate the Biological Effects and Safety of a Polyphenol Supplement on Healthy Ageing.

DailyColors™ is a supplement made up of several phytonutrients that aims to replicate elements from the Mediterranean diet. These include fruit, berry and vegetable extracts that are rich in key phytochemicals such as Quercetin, Catechins, Phloretin, Ellagic Acid, and Anthocyanins. Here, we determined the effects of DailyColors™ on the blood biomarkers associated with the diverse mechanisms implicated in ageing and age-related diseases, including mitochondrial function, inflammation, and oxidative stress, as well as on saliva's DNA methylation pattern. Thirty adult participants (mean (SD) age = 67.0 (7.5) years) with a body mass index over 25 were recruited into this randomised, double-blind, placebo-controlled, cross-over trial (two one-week treatment periods, separated by a one-week washout period). During the placebo period, we observed a significant increase in blood CD38 concentrations from the baseline to 24 h (p-value = 0.019). This was not observed in the active period. Increased CD38 is reportedly associated with subsequent mitochondrial dysfunction and inflammation. Next, there was a decreasing trend of plasma 4-HNE levels, an oxidative stress biomarker, after a one-week intake of DailyColors™. Furthermore, following a one-month open-label follow-up in 26 participants, we observed hypermethylation of the candidate CpG site cg13108341 (q-value = 0.021), which was against the observed trend for this site during ageing. Taken together, while minimal effects were observed in this study, DailyColors™ supplementation may be beneficial by altering and alleviating age-related changes. Longer and larger scale trials of DailyColors™ supplementation are warranted.

A randomized, placebo-controlled trial of purified anthocyanins on cognitive function in individuals at elevated risk for dementia: Analysis of inflammatory biomarkers toward personalized interventions.

BACKGROUND: Dementia poses a significant global health challenge. Anthocyanins neutralize free radicals, modulate signaling pathways, inhibit pro-inflammatory genes, and suppress cytokine production and may thus have positive cognitive effects in people at increased risk of dementia. We aim to investigate the effects of purified anthocyanins on cognitive function in people at increased risk of dementia according to their inflammation status based on blood-based inflammatory biomarkers. METHODS: This is a secondary analysis of a 24-week randomized, double-blind, placebo-controlled trial. Cluster analysis was performed to categorize two groups based on their individual inflammatory biomarker profile using multiplex sandwich ELISA for the quantitative measurement of cytokines. Descriptive statistics and longitudinal models assessed cognitive outcomes. The primary comparison was the group difference at week 24 based on a modified intention-to-treat analysis. RESULTS: Cluster analysis revealed two distinct inflammatory biomarker profiles. In Cluster 1 (high levels of inflammation biomarkers), anthocyanin treatment showed a statistically significant improvement on cognitive function compared to placebo at 24 weeks. No significant differences were observed in Cluster 2 (low levels of inflammation biomarkers). The demographic characteristics, cognitive scores, and biomarker distributions were similar between treatment groups at baseline. However, cluster 1 exhibited higher BMI, diabetes prevalence, medication usage, and lower HDL cholesterol levels. CONCLUSION: Individuals with elevated levels of inflammation markers benefited from anthocyanin treatment to enhance cognitive performance, whereas those with lower levels did not. The anti-inflammatory and antioxidant properties of anthocyanins make them a promising intervention, and future prospective trials in people with increased inflammation are warranted.

Global, regional, and national burden of HIV and other sexually transmitted infections in older adults aged 60-89 years from 1990 to 2019: results from the Global Burden of Disease Study 2019.

BACKGROUND: Sexually active older adults are often more susceptible to HIV and other sexually transmitted infections (STIs) due to various health conditions (especially a weakened immune system) and low use of condoms. We aimed to assess the global, regional, and national burdens and trends of HIV and other STIs in older adults from 1990 to 2019. METHODS: We retrieved data from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 on the incidence and disability-adjusted life-years (DALYs) of HIV and other STIs (syphilis, chlamydia, gonorrhoea, trichomoniasis, and genital herpes) for older adults aged 60-89 years in 204 countries and territories from 1990 to 2019. Estimated annual percentage changes in the age-standardised incidence and DALY rates of HIV and other STIs, by age, sex, and Socio-demographic Index (SDI), were calculated to quantify the temporal trends. Spearman correlation analysis was used to examine the relationship between age-standardised rates and SDI. FINDINGS: In 2019, among older adults globally, there were an estimated 77 327 (95% uncertainty interval 59 443 to 97 648) new cases of HIV (age-standardised incidence rate 7·6 [5·9 to 9·6] per 100 000 population) and 26 414 267 (19 777 666 to 34 860 678) new cases of other STIs (2607·1 [1952·1 to 3440·8] per 100 000). The age-standardised incidence rate decreased by an average of 2·02% per year (95% CI -2·38 to -1·66) for HIV and remained stable for other STIs (-0·02% [-0·06 to 0·01]) from 1990 to 2019. The number of DALYs globally in 2019 was 1 905 099 (95% UI 1 670 056 to 2 242 807) for HIV and 132 033 (95% UI 83 512 to 225 630) for the other STIs. The age-standardised DALY rate remained stable from 1990 to 2019, with an average change of 0·97% (95% CI -0·54 to 2·50) per year globally for HIV but decreased by an annual average of 1·55% (95% CI -1·66 to -1·43) for other STIs. Despite the global decrease in the age-standardised incidence rate of HIV in older people from 1990 to 2019, many regions showed increases, with the largest increases seen in eastern Europe (average annual change 17·84% [14·16 to 21·63], central Asia (14·26% [11·35 to 17·25]), and high-income Asia Pacific (7·52% [6·54 to 8·51]). Regionally, the age-standardised incidence and DALY rates of HIV and other STIs decreased with increases in the SDI. INTERPRETATION: Although the incidence and DALY rates of HIV and STIs either declined or remained stable from 1990 to 2019, there were regional and demographic disparities. Health-care providers should be aware of the effects of ageing societies and other societal factors on the risk of HIV and other STIs in older adults, and develop age-appropriate interventions. The disparities in the allocation of health-care resources for older adults among regions of different SDIs should be addressed. FUNDING: Natural Science Foundation of China, Fujian Province's Third Batch of Flexible Introduction of High-Level Medical Talent Teams, Science and Technology Innovation Team (Tianshan Innovation Team) Project of Xinjiang Uighur Autonomous Region, Cure Alzheimer's Fund, Helse Sør-Øst, the Research Council of Norway, Molecule/VitaDAO, NordForsk Foundation, Akershus University Hospital, the Civitan Norges Forskningsfond for Alzheimers Sykdom, the Czech Republic-Norway KAPPA programme, and the Rosa Sløyfe/Norwegian Cancer Society & Norwegian Breast Cancer Society.

Temporal and spatial distribution of Nrf2 in rat brain following stroke: quantification of nuclear to cytoplasmic Nrf2 content using a novel immunohistochemical technique.

Activation of the redox-sensitive transcription factor NF-E2 related factor 2 (Nrf2) affords protection against cerebral ischaemia-reperfusion injury via the upregulation of antioxidant defence genes. We have quantified for the first time Nrf2 content in brains from rats subjected to stroke and from cultured bEnd.3 brain endothelial cells using a novel immunohistochemical technique. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 70 min followed by reperfusion for 4, 24 or 72 h. Coronal brain sections were incubated with anti-Nrf2 primary and biotinylated-horseradish peroxidase-conjugated secondary antibody, after which sections were reacted with 3,3-diaminobenzidine (DAB) in the presence of hydrogen peroxide. The initial rates of DAB polymer formation were directly proportional to the Nrf2 protein concentration. Image processing was used to determine the temporal and spatial distribution of Nrf2 in nuclear and cytoplasmic compartments in stroke-affected and contralateral hemispheres. Nuclear to cytoplasmic Nrf2 ratios were increased in the stroke region after 24 h reperfusion and declined after 72 h reperfusion. Pretreatment with the Nrf2 inducer sulforaphane reduced total cellular Nrf2 levels in peri-infarct and core regions of the stroke hemisphere after 24 h reperfusion. Treatment of cultured murine brain endothelial cells with sulforaphane (2.5 µm) increased nuclear accumulation of Nrf2 over 1-4 h. We report the first quantitative measurements of spatial and temporal nuclear Nrf2 expression in rat brains following stroke, and show that sulforaphane pretreatment affects Nrf2 distribution in the brain of naïve rats and animals subjected to cerebral ischaemia. Our findings provide novel insights for targeting endogenous redox-sensitive antioxidant pathways to ameliorate the damaging consequences of stroke.

Reduced Levels of the Antiaging Hormone Klotho are Associated With Increased Aortic Stiffness in Diabetic Kidney Disease.

Introduction: Aortic pulse wave velocity (Ao-PWV) predicts cardiovascular and kidney disease in type 2 diabetes (T2D). Klotho is a circulating antiaging hormone (sKlotho) with putative cardiorenal protective effects. The relationship between sKlotho and Ao-PWV in diabetic kidney disease (DKD) is unknown. Methods: In a cross-sectional cohort study, the correlation of sKlotho measured by a validated immunoassay, and Ao-PWV measured by applanation tonometry, was investigated in 172 participants with T2D and early stage DKD (all had estimated glomerular filtration rate [eGFR] >45 ml/min) on stable renin angiotensin system (RAS) inhibition. In cultured human aortic smooth muscle cells (HASMCs) stimulated with angiotensin II (AngII), the effects of recombinant human sKlotho pretreatment were assessed on intracellular calcium ([Ca2+]i) responses and expression of proteins associated with proosteogenic HASMC phenotypes. Results: Mean (range) age of the cohort was 61.3 years (40-82) and 65% were male. Mean (±SD) Ao-PWV was 11.4 (±2.3) m/s, eGFR 78.8 (±23.5) and median (interquartile range) sKlotho of 358.5 (194.2-706.3) pg/ml. In multivariable linear regression analyses, we observed a statistically significant inverse relationship between sKlotho and Ao-PWV, which was independent of clinical risk factors for cardiorenal disease. Pretreatment of cultured HASMC with sKlotho significantly attenuated AngII-stimulated [Ca2+]i transients and reduced osteogenic collagen (Col1a2) expression. Conclusions: In individuals with T2D and early DKD, lower levels of sKlotho are associated with increased Ao-PWV. Taken together with the direct effect of sKlotho on mediators of aortic wall stiffness in vitro, these findings may explain the enhanced risk of cardiorenal disease in DKD.

Redox and metal profiles in human coronary endothelial and smooth muscle cells under hyperoxia, physiological normoxia and hypoxia: Effects of NRF2 signaling on intracellular zinc.

Zinc is an important component of cellular antioxidant defenses and dysregulation of zinc homeostasis is a risk factor for coronary heart disease and ischemia/reperfusion injury. Intracellular homeostasis of metals, such as zinc, iron and calcium are interrelated with cellular responses to oxidative stress. Most cells experience significantly lower oxygen levels in vivo (2-10 kPa O2) compared to standard in vitro cell culture (18kPa O2). We report the first evidence that total intracellular zinc content decreases significantly in human coronary artery endothelial cells (HCAEC), but not in human coronary artery smooth muscle cells (HCASMC), after lowering of O2 levels from hyperoxia (18 kPa O2) to physiological normoxia (5 kPa O2) and hypoxia (1 kPa O2). This was paralleled by O2-dependent differences in redox phenotype based on measurements of glutathione, ATP and NRF2-targeted protein expression in HCAEC and HCASMC. NRF2-induced NQO1 expression was attenuated in both HCAEC and HCASMC under 5 kPa O2 compared to 18 kPa O2. Expression of the zinc efflux transporter ZnT1 increased in HCAEC under 5 kPa O2, whilst expression of the zinc-binding protein metallothionine (MT) decreased as O2 levels were lowered from 18 to 1 kPa O2. Negligible changes in ZnT1 and MT expression were observed in HCASMC. Silencing NRF2 transcription reduced total intracellular zinc under 18 kPa O2 in HCAEC with negligible changes in HCASMC, whilst NRF2 activation or overexpression increased zinc content in HCAEC, but not HCASMC, under 5 kPa O2. This study has identified cell type specific changes in the redox phenotype and metal profile in human coronary artery cells under physiological O2 levels. Our findings provide novel insights into the effect of NRF2 signaling on Zn content and may inform targeted therapies for cardiovascular diseases.

Vascular protection afforded by zinc supplementation in human coronary artery smooth muscle cells mediated by NRF2 signaling under hypoxia/reoxygenation.

Zinc (Zn) has antioxidant, anti-inflammatory and anti-proliferative actions, with Zn dysregulation associated with coronary ischemia/reperfusion injury and smooth muscle cell dysfunction. As the majority of studies concerning Zn have been conducted under non-physiological hyperoxic conditions, we compare the effects of Zn chelation or supplementation on total intracellular Zn content, antioxidant NRF2 targeted gene transcription and hypoxia/reoxygenation-induced reactive oxygen species generation in human coronary artery smooth muscle cells (HCASMC) pre-adapted to hyperoxia (18 kPa O2) or normoxia (5 kPa O2). Expression of the smooth muscle marker SM22-α was unaffected by lowering pericellular O2, whereas calponin-1 was significantly upregulated in cells under 5 kPa O2, indicating a more physiological contractile phenotype under 5 kPa O2. Inductively coupled plasma mass spectrometry established that Zn supplementation (10 µM ZnCl2 + 0.5 µM pyrithione) significantly increased total Zn content in HCASMC under 18 but not 5 kPa O2. Zn supplementation increased metallothionein mRNA expression and NRF2 nuclear accumulation in cells under 18 or 5 kPa O2. Notably, NRF2 regulated HO-1 and NQO1 mRNA expression in response to Zn supplementation was only upregulated in cells under 18 but not 5 kPa. Furthermore, whilst hypoxia increased intracellular glutathione (GSH) in cells pre-adapted to 18 but not 5 kPa O2, reoxygenation had negligible effects on GSH or total Zn content. Reoxygenation-induced superoxide generation in cells under 18 kPa O2 was abrogated by PEG-superoxide dismutase but not by PEG-catalase, and Zn supplementation, but not Zn chelation, attenuated reoxygenation-induced superoxide generation in cells under 18 but not 5kPaO2, consistent with a lower redox stress under physiological normoxia. Our findings highlight that culture of HCASMC under physiological normoxia recapitulates an in vivo contractile phenotype and that effects of Zn on NRF2 signaling are altered by oxygen tension.

Spotlight issue: MicroRNAs in the microcirculation--from cellular mechanisms to clinical markers.

This spotlight issue of Microcirculation contains four state-of-the-art review articles on the role of microRNAs (miRNAs), a class of endogenous, highly conserved, small, non-coding RNAs that regulate gene expression at the post transcriptional level, and can act as key regulators of cellular mechanisms within the microcirculation. The expert reviews address issues, such as the role of miRNAs in determining endothelial cell differentiation and lineage commitment, the physiological role of miRNAs as critical modulators of endothelial cell proliferation, apoptosis and in angiogenesis, and their aberrant expression in different vascular disorders. The reviews also explore the prognostic value of miRNAs in cardiovascular disease and how they may serve both as a therapeutic target and clinical biomarker in the future. This cutting edge edition of the journal Microcirculation highlights the progress that has been made in this new and challenging research area.

Targeting the Nrf2-Keap1 antioxidant defence pathway for neurovascular protection in stroke.

Endogenous defence mechanisms by which the brain protects itself against noxious stimuli and recovers from ischaemic damage are a key target of stroke research. The loss of viable brain tissue in the ischaemic core region after stroke is associated with damage to the surrounding area known as the penumbra. Activation of the redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) plays a pivotal role in the cellular defence against oxidative stress via transcriptional upregulation of phase II defence enzymes and antioxidant stress proteins. Although recent evidence implicates Nrf2 in neuroprotection, it is not known whether activation of this pathway within the neurovascular unit protects the brain against blood-brain barrier breakdown and cerebrovascular inflammation. Targeting the neurovascular unit should provide novel insights for effective treatment strategies and facilitate translation of experimental findings into clinical therapy. This review focuses on the cytoprotective role of Nrf2 in stroke and examines the evidence that the Nrf2-Keap1 defence pathway may serve as a therapeutic target for neurovascular protection.

Impact of dietary soy isoflavones in pregnancy on fetal programming of endothelial function in offspring.

Epidemiological evidence suggests that soy-based diets containing phytoestrogens (isoflavones) afford protection against cardiovascular diseases (CVDs); however, supplementation trials have largely reported only marginal health benefits. The molecular mechanisms by which the isoflavones genistein, daidzein, and equol afford protection against oxidative stress remain to be investigated in large scale clinical trials. Isoflavones are transferred across the placenta in both rodents and humans, yet there is limited information on their actions in pregnancy and the developmental origins of disease. Our studies established that feeding a soy isoflavone-rich diet during pregnancy, weaning, and postweaning affords cardiovascular protection in aged male rats. Notably, rats exposed to a soy isoflavone-deficient diet throughout pregnancy and adult life exhibited increased oxidative stress, diminished antioxidant enzyme and eNOS levels, endothelial dysfunction, and elevated blood pressure in vivo. The beneficial effects of refeeding isoflavones to isoflavone-deficient rats include an increased production of nitric oxide and EDHF, an upregulation of antioxidant defense enzymes and lowering of blood pressure in vivo. This review focuses on the role that isoflavones in the fetal circulation may play during fetal development in affording protection against CVD in the offspring via their ability to activate eNOS, EDHF, and redox-sensitive gene expression.

The potential of rapalogs to enhance resilience against SARS-CoV-2 infection and reduce the severity of COVID-19.

COVID-19 disproportionately affects older people, with likelihood of severe complications and death mirroring that of other age-associated diseases. Inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) has been shown to delay or reverse many age-related phenotypes, including declining immune function. Rapamycin (sirolimus) and rapamycin derivatives are US Food and Drug Administration-approved inhibitors of mTORC1 with broad clinical utility and well established dosing and safety profiles. Based on preclinical and clinical evidence, a strong case can be made for immediate large-scale clinical trials to assess whether rapamycin and other mTORC1 inhibitors can prevent COVID-19 infection in these populations and also to determine whether these drugs can improve outcomes in patients with severe COVID-19.

Ageing modulates human dermal fibroblast contractility: Quantification using nano-biomechanical testing.

Dermal fibroblasts play a key role in maintaining homoeostasis and functionality of the skin. Their contractility plays a role in changes observed during ageing, especially in processes such as wound healing, inflammation, wrinkling and scar tissue formation as well as structural changes on extracellular matrix. Although alternations in skin physiology and morphology have been previously described, there remains a paucity of information about the influence of chronological ageing on dermal fibroblast contractility. In this study, we applied a novel nano-biomechanical technique on cell-embedded collagen hydrogels in combination with mathematical modelling and numerical simulation to measure contraction forces of normal human dermal fibroblasts (NHDF). We achieved quantitative differentiation of the contractility of cells derived from 'young' (< 30 years old) and 'aged' (> 60 years old) donors. Transforming growth factor ß1 (TGF-ß1) was used to stimulate the fibroblasts to assess their contractile potential. NHDF from aged donors exhibited a greater basal contractile force, while in contrast, NHDF from young donors have shown a significantly larger contractile force in response to TGF-ß1 treatment. These findings validate our nano-biomechanical measurement technique and provide new insights for considering NHDF contractility in regenerative medicine and as a biomarker of dermal ageing processes.