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.

Meeting Summary of The NYO3 5th NO-Age/AD Meeting and the 1st Norway-UK Joint Meeting on Aging and Dementia: Recent Progress on the Mechanisms and Interventional Strategies.

Unhealthy aging poses a global challenge with profound healthcare and socioeconomic implications. Slowing down the aging process offers a promising approach to reduce the burden of a number of age-related diseases, such as dementia, and promoting healthy longevity in the old population. In response to the challenge of the aging population and with a view to the future, Norway and the United Kingdom are fostering collaborations, supported by a "Money Follows Cooperation agreement" between the 2 nations. The inaugural Norway-UK joint meeting on aging and dementia gathered leading experts on aging and dementia from the 2 nations to share their latest discoveries in related fields. Since aging is an international challenge, and to foster collaborations, we also invited leading scholars from 11 additional countries to join this event. This report provides a summary of the conference, highlighting recent progress on molecular aging mechanisms, genetic risk factors, DNA damage and repair, mitophagy, autophagy, as well as progress on a series of clinical trials (eg, using NAD+ precursors). The meeting facilitated dialogue among policymakers, administrative leaders, researchers, and clinical experts, aiming to promote international research collaborations and to translate findings into clinical applications and interventions to advance healthy aging.

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.

HTATIP2 regulates arteriogenic activity in monocytes from patients with limb ischemia.

Use of autologous cells isolated from elderly patients with multiple comorbidities may account for the modest efficacy of cell therapy in patients with chronic limb threatening ischemia (CLTI). We aimed to determine whether proarteriogenic monocyte/macrophages (Mo/MΦs) from patients with CLTI were functionally impaired and to demonstrate the mechanisms related to any impairment. Proarteriogenic Mo/MΦs isolated from patients with CLTI were found to have an impaired capacity to promote neovascularization in vitro and in vivo compared with those isolated from healthy controls. This was associated with increased expression of human HIV-1 TAT interactive protein-2 (HTATIP2), a transcription factor known to suppress angiogenesis/arteriogenesis. Silencing HTATIP2 restored the functional capacity of CLTI Mo/MΦs, which was associated with increased expression of arteriogenic regulators Neuropilin-1 and Angiopoietin-1, and their ability to enhance angiogenic (endothelial tubule formation) and arteriogenic (smooth muscle proliferation) processes in vitro. In support of the translational relevance of our findings, silencing HTATIP2 in proarteriogenic Mo/MΦs isolated from patients with CLTI rescued their capacity to enhance limb perfusion in the ischemic hindlimb by effecting greater angiogenesis and arteriogenesis. Ex vivo modulation of HTATIP2 may offer a strategy for rescuing the functional impairment of pro-angio/arteriogenic Mo/MΦs prior to autologous delivery and increase the likelihood of clinical efficacy.

Tracing the transmission of mpox through wastewater surveillance in Southeast Asia.

High population density and tourism in Southeast Asia increase the risk of mpox due to frequent interpersonal contacts. Our wastewater surveillance in six Southeast Asian countries revealed positive signals for Monkeypox virus (MPXV) DNA, indicating local transmission. This alerts clinicians and helps allocate resources like testing, vaccines and therapeutics in resource-limited countries.

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.

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.

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.

Sexual dimorphism in COVID-19: potential clinical and public health implications.

Current evidence suggests that severity and mortality of COVID-19 is higher in men than in women, whereas women might be at increased risk of COVID-19 reinfection and development of long COVID. Differences between sexes have been observed in other infectious diseases and in the response to vaccines. Sex-specific expression patterns of proteins mediating virus binding and entry, and divergent reactions of the immune and endocrine system, in particular the hypothalamic-pituitary-adrenal axis, in response to acute stress might explain the higher severity of COVID-19 in men. In this Personal View, we discuss how sex hormones, comorbidities, and the sex chromosome complement influence these mechanisms in the context of COVID-19. Due to its role in the severity and progression of SARS-CoV-2 infections, we argue that sexual dimorphism has potential implications for disease treatment, public health measures, and follow-up of patients predisposed to the development of long COVID. We suggest that sex differences could be considered in future pandemic surveillance and treatment of patients with COVID-19 to help to achieve better disease stratification and improved outcomes.

Corrigendum: Effects of Anthocyanin Supplementation on Serum Lipids, Glucose, Markers of Inflammation and Cognition in Adults With Increased Risk of Dementia - A Pilot Study.

[This corrects the article DOI: 10.3389/fgene.2019.00536.].

Differential Expression of Insulin-Like Growth Factor 1 and Wnt Family Member 4 Correlates With Functional Heterogeneity of Human Dermal Fibroblasts.

Although human dermis contains distinct fibroblast subpopulations, the functional heterogeneity of fibroblast lines from different donors is under-appreciated. We identified one commercially sourced fibroblast line (c64a) that failed to express α-smooth muscle actin (α-SMA), a marker linked to fibroblast contractility, even when treated with transforming growth factor-ß1 (TGF-ß1). Gene expression profiling identified insulin-like growth factor 1 (IGF1) as being expressed more highly, and Asporin (ASPN) and Wnt family member 4 (WNT4) expressed at lower levels, in c64a fibroblasts compared to three fibroblast lines that had been generated in-house, independent of TGF-ß1 treatment. TGF-ß1 increased expression of C-X-C motif chemokine ligand 1 (CXCL1) in c64a cells to a greater extent than in the other lines. The c64a gene expression profile did not correspond to any dermal fibroblast subpopulation identified by single-cell RNAseq of freshly isolated human skin cells. In skin reconstitution assays, c64a fibroblasts did not support epidermal stratification as effectively as other lines tested. In fibroblast lines generated in-house, shRNA-mediated knockdown of IGF1 increased α-SMA expression without affecting epidermal stratification. Conversely, WNT4 knockdown had no consistent effect on α-SMA expression, but increased the ability of fibroblasts to support epidermal stratification. Thus, by comparing the properties of different lines of cultured dermal fibroblasts, we have identified IGF1 and WNT4 as candidate mediators of two distinct dermal functions: myofibroblast formation and epidermal maintenance.

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.

Advances in microfluidic in vitro systems for neurological disease modeling.

Neurological disorders are the leading cause of disability and the second largest cause of death worldwide. Despite significant research efforts, neurology remains one of the most failure-prone areas of drug development. The complexity of the human brain, boundaries to examining the brain directly in vivo, and the significant evolutionary gap between animal models and humans, all serve to hamper translational success. Recent advances in microfluidic in vitro models have provided new opportunities to study human cells with enhanced physiological relevance. The ability to precisely micro-engineer cell-scale architecture, tailoring form and function, has allowed for detailed dissection of cell biology using microphysiological systems (MPS) of varying complexities from single cell systems to "Organ-on-chip" models. Simplified neuronal networks have allowed for unique insights into neuronal transport and neurogenesis, while more complex 3D heterotypic cellular models such as neurovascular unit mimetics and "Organ-on-chip" systems have enabled new understanding of metabolic coupling and blood-brain barrier transport. These systems are now being developed beyond MPS toward disease specific micro-pathophysiological systems, moving from "Organ-on-chip" to "Disease-on-chip." This review gives an outline of current state of the art in microfluidic technologies for neurological disease research, discussing the challenges and limitations while highlighting the benefits and potential of integrating technologies. We provide examples of where such toolsets have enabled novel insights and how these technologies may empower future investigation into neurological diseases.

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.

Glycocalyx sialic acids regulate Nrf2-mediated signaling by fluid shear stress in human endothelial cells.

Activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is critical for vascular endothelial redox homeostasis in regions of high, unidirectional shear stress (USS), however the underlying mechanosensitive mediators are not fully understood. The endothelial glycocalyx is disrupted in arterial areas exposed to disturbed blood flow that also exhibit enhanced oxidative stress leading to atherogenesis. We investigated the contribution of glycocalyx sialic acids (SIA) to Nrf2 signaling in human endothelial cells (EC) exposed to atheroprotective USS or atherogenic low oscillatory shear stress (OSS). Cells exposed to USS exhibited a thicker glycocalyx and enhanced turnover of SIA which was reduced in cells cultured under OSS. Physiological USS, but not disturbed OSS, enhanced Nrf2-mediated expression of antioxidant enzymes, which was attenuated following SIA cleavage with exogenous neuraminidase. SIA removal disrupted kinase signaling involved in the nuclear accumulation of Nrf2 elicited by USS and promoted mitochondrial reactive oxygen species accumulation. Notably, knockdown of the endogenous sialidase NEU1 potentiated Nrf2 target gene expression, directly implicating SIA in regulation of Nrf2 signaling by USS. In the absence of SIA, deficits in Nrf2 responses to physiological flow were also associated with a pro-inflammatory EC phenotype. This study demonstrates that the glycocalyx modulates endothelial redox state in response to shear stress and provides the first evidence of an atheroprotective synergism between SIA and Nrf2 antioxidant signaling. The endothelial glycocalyx therefore represents a potential therapeutic target against EC dysfunction in cardiovascular disease and redox dyshomeostasis in ageing.

Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation.

Ischemic stroke is associated with a surge in reactive oxygen species generation during reperfusion. The narrow therapeutic window for the delivery of intravenous thrombolysis and endovascular thrombectomy limits therapeutic options for patients. Thus, understanding the mechanisms regulating neurovascular redox defenses are key for improved clinical translation. Our previous studies in a rodent model of ischemic stroke established that activation of Nrf2 defense enzymes by pretreatment with sulforaphane (SFN) affords protection against neurovascular and neurological deficits. We here further investigate SFN mediated protection in mouse brain microvascular endothelial cells (bEnd.3) adapted long-term (5 days) to hyperoxic (18 kPa) and normoxic (5 kPa) O2 levels. Using an O2-sensitive phosphorescent nanoparticle probe, we measured an intracellular O2 level of 3.4 ± 0.1 kPa in bEnd 3 cells cultured under 5 kPa O2. Induction of HO-1 and GCLM by SFN (2.5 µM) was significantly attenuated in cells adapted to 5 kPa O2, despite nuclear accumulation of Nrf2. To simulate ischemic stroke, bEnd.3 cells were adapted to 18 or 5 kPa O2 and subjected to hypoxia (1 kPa O2, 1 h) and reoxygenation. In cells adapted to 18 kPa O2, reoxygenation induced free radical generation was abrogated by PEG-SOD and significantly attenuated by pretreatment with SFN (2.5 µM). Silencing Nrf2 transcription abrogated HO-1 and NQO1 induction and led to a significant increase in reoxygenation induced free radical generation. Notably, reoxygenation induced oxidative stress, assayed using the luminescence probe L-012 and fluorescence probes MitoSOX™ Red and FeRhoNox™-1, was diminished in cells cultured under 5 kPa O2, indicating an altered redox phenotype in brain microvascular cells adapted to physiological normoxia. As redox and other intracellular signaling pathways are critically affected by O2, the development of antioxidant therapies targeting the Keap1-Nrf2 defense pathway in treatment of ischemia-reperfusion injury in stroke, coronary and renal disease will require in vitro studies conducted under well-defined O2 levels.