Methodological evaluation of muscle oxidative stress in normotensive and hypertensive individuals: Ageing‐related

Because arterial hypertension (AH) is a significant global health issue affecting almost 900 million people worldwide, researchers have been dedicated to understanding its pathogenesis and refining methodological evaluations to propose therapeutic approaches (Oparil et al., 2018). Annually, non-optimal blood pressure accounts for ∼9.4 million deaths worldwide. This evidence underscores the urgent need for effective public health strategies to mitigate the impact of AH and its associated complications on global health. The pathophysiology of AH is multifaceted, involving several cellular and molecular mechanisms. Some primary pathways have been extensively studied, including the renin–angiotensin–aldosterone system, inflammation, oxidative stress and extracellular matrix remodelling (Frąk et al., 2022; Oparil et al., 2018). Nonetheless, the incidence of AH in ageing populations highlights the necessity of reassessing the vascular mechanisms underlying age-related blood pressure changes (Baksi et al., 2009). In their article published in The Journal of Physiology, Speer et al. (2024) describe their project aimed at characterizing microvascular health and functional changes in skeletal muscle among normotensive and hypertensive middle-aged to older adults, independent of poor lifestyle factors and comorbidities. Their outcomes will provide novel insights into the systemic ramifications of AH. Speer et al. (2024) propose that microvascular disturbances serve as early biomarkers of systemic haemodynamic pathology, emphasizing the importance of early detection and prevention strategies. The collaborators focus on endothelial dysfunction induced by oxidative stress in the skeletal muscle microcirculation, which leads to reduced nitric oxide (NO) bioavailability, impairing vasodilatory responses and contributing to increased vascular stiffness, which is a core feature of haemodynamic ageing and a precursor to AH (Speer et al., 2024). Oxidant-antioxidant imbalance plays a crucial role in the cardiovascular system, driven primarily by NADPH oxidases (NOX), particularly NOX1, which are stimulated by interferon, interleukin-1 and angiotensin II (Manea et al., 2015). This leads to an imbalance between reactive oxygen species (ROS) production and antioxidant defences. Interestingly, a heightened pro-oxidant state itself influences the expression of nuclear factor erythroid 2-related factor 2, a major regulator of antioxidant responses and cytoprotective genes (e.g. superoxide dismutase), ultimately decreasing the body's ability to counteract ROS accumulation (Frąk et al., 2022). Additionally, oxidative burden promotes vascular smooth muscle cell proliferation, contributing to the recruitment of inflammatory cells within the vasculature via the chaperone protein cyclophilin A, which stimulates matrix metalloproteinases (MMPs) (Frąk et al., 2022). MMPs, along with their tissue inhibitors, orchestrate extracellular matrix components such as collagen, elastin, laminin and proteoglycans. Excessive ROS contribute to decreased NO bioavailability, which is an important vasodilator. Increased levels of pro-inflammatory cytokines, NOX and MMPs, as well as decreased redox homeostasis and NO, have been associated with hypertensive conditions (Frąk et al., 2022; Oparil et al., 2018). Other contributors also exacerbate endothelial injury and inflammation (Frąk et al., 2022; Oparil et al., 2018). Ultimately, these interconnected mechanisms compromise endothelial function, leading to increased vascular resistance and lack of elasticity reflecting systemic circulatory complications. The onset of these complex mechanisms is often attributed to poor lifestyle habits (e.g. inadequate nutrition, physical inactivity and substance abuse). Thus, it is necessary to elucidate whether these mechanisms are also involved in the AH linked with the progression of senescence (Figure 1). To investigate ROS dynamics and microvascular function in skeletal muscle, Speer et al. (2024) have implemented a novel approach utilizing muscle microdialysis, representing a significant methodological innovation in this area. This technique involves the insertion of microdialysis probes into the vastus lateralis muscle under local anaesthesia. Two probes, positioned ∼3 cm apart, are perfused with a saline–ethanol solution at a controlled rate, allowing the muscle tissue to stabilize. ROS production is assessed by perfusing the probes with a solution containing Amplex UltraRed (Thermo Fisher Scientific, Waltham, MA, USA) and horseradish peroxidase to detect hydrogen peroxide (H₂O₂) formation. In a subsequent phase, superoxide dismutase is introduced into the perfusate, facilitating the conversion of superoxide anions (O₂−) to H₂O₂. The differential signal between these two phases provides a quantitative measure of in vivo O₂− generation. Microvascular function has been assessed through endothelium-dependent vasodilatation via the administration of ACh in the perfusate. The study ensures technical rigour by alternating syringe contents between probes across different experimental phases, with sample collection at 15 min intervals for immediate laboratory analysis. Nutritive blood flow has been inferred from ethanol outflow-to-inflow ratios, where higher ratios indicate reduced local blood flow. The ACh-induced modulation of these ratios serves as a functional measure of endothelial-dependent vasodilatation. This methodological approach offers real-time insights into muscle ROS dynamics and microvascular function, bridging the gap between systemic human studies and in vitro investigations. Unlike traditional techniques requiring multiple invasive procedures, this strategy simultaneously evaluates ROS levels, endothelial function and local blood flow within a single clinical session, enhancing its translational relevance. The initial findings indicate that these procedures are viable and can be continually implemented (Speer et al., 2024). The research employs a rigorous experimental design in which participants are carefully selected to minimize confounding factors such as metabolic disorders and antioxidant supplementation. The protocol includes assessments of aerobic fitness, dietary intake, activity exercise questionnaire and body composition, providing a comprehensive evaluation of each participant's health (Speer et al., 2024). The researchers endeavour to identify vascular changes attributable primarily to age-related AH. Yet, some limitations remain. Oparil et al. (2018) have referenced studies conducted on isolated societies with minimal exposure to external influences, suggesting that blood pressure elevation in ageing individuals predominantly occurs following migration to urban environments, where lifestyle factors deteriorate. These data reinforce the critical role of social, environmental and lifestyle modifications in the progression of AH and introduce substantial variability that may act as a confounding factor, potentially obscuring the direct contributions of senescence to circulatory dysfunction. Hence, a fundamental limitation in testing the impact of ageing on high blood pressure is the cumulative influence of an individual's medical history on their cardiovascular health across life stages. Factors such as childhood obesity, prolonged periods of physical inactivity, past smoking habits and other historical conditions that may have previously compromised endothelial function, despite no longer being active contributors, might pose barriers to segregating the direct effects of ageing. To enhance the precision of research on blood pressure disorders associated with ageing, it is recommended that investigators conduct a comprehensive and systematic assessment of participants' medical histories. This can be achieved through the integration of validated screening instruments, such as the History of Diseases and Infections Questionnaire, the Alcohol, Smoking and Substance Involvement Screening Test (ASSIST), and the Weight History Questionnaire, or the creation of a specific tool tailored for this type of investigation. Employing such tools would enable researchers to more effectively differentiate the intrinsic endothelial effects of ageing from those attributable to prior lifestyle and environmental exposures, thereby optimizing the accuracy of conclusions drawn regarding senescence-driven AH. In conclusion, the study by Speer et al. (2024) significantly contributes to advancing the field by leveraging innovative methodologies that pave the way for a deeper comprehension of the relationship between ROS production and microvascular function in the population. However, despite the implementation of rigorous methodologies, fully isolating the influence of ageing remains a considerable challenge. Beyond incorporating a comprehensive lifespan health history questionnaire, future research directions could explore NOX levels, MMP regulation and immune system interactions to further refine our understanding of age-related hypertension and distinguish it from elevated blood pressure induced by comorbidities. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. None declared. All authors have approved the final manuscript for publication and take full responsibility for its content. Both authors meet the authorship criteria. This work was supported by the Coordination for the Improvement of Higher Education Personnel (CAPES; finance code 001) and by the São Paulo Research Foundation (FAPESP; Process number: 2022/60 807-2).