The role of transient receptor potential ankyrin 1 in age-related endothelial dysfunction
Introduction
Endothelial dysfunction is defined as reduced endothelium-dependent vascular dilation in response to chemical or mechanical stimuli, which is characterized by decreased relaxing factors and increased contracting factors released by the endothelium (Deanfield et al., 2007). Nitric oxide (NO), produced by endothelial NO synthase (eNOS), is the most important endothelium-derived relaxing factor (Murata et al., 2002). Reduced bioavailability of NO results from decreased eNOS activity and/or increased oxidative stress with excessive reactive oxygen species (ROS) such as superoxide that can quench the bioactivity of NO (Forstermann and Munzel, 2006). Preclinical and clinical studies demonstrated that aging is an important risk factor for endothelial dysfunction, which is associated with an increased risk of cardiovascular disease in the elderly population (Ungvari et al., 2018). Enhanced oxidative stress and decreased NO bioavailability are considered the key mechanisms mediating age-related endothelial dysfunction (Herrera et al., 2010). Oxidative stress also contributes to replicative senescence (Barbouti et al., 2020). Given an increasing aging population and increased risk of cardiovascular disease in older adults, effective strategies are needed to prevent and treat age-related endothelial dysfunction (Seals et al., 2011).
The transient receptor potential ankyrin subtype 1 (TRPA1) channel is a nonselective cation channel expressed by afferent nociceptive neurons and non-excitable cells such as endothelial cells (Earley et al., 2009). TRPA1 that has cysteine residues in the cytoplasmic N terminus can be activated by environmental pungent chemicals and oxidative stress byproducts such as superoxide (Xu et al., 2019). TRPA1 acts as a sensor of oxidative stress and is involved in an oxidative-stress defense mechanism (Takahashi et al., 2018). Previous studies demonstrated that TRPA1 promoted longevity of C. elegans and that activating TRPA1 rescued α-dicarbonyls-induced aging-related pathologies in C. elegans (Chaudhuri et al., 2016; Xiao et al., 2013; Zhang et al., 2015). However, the role of TRPA1 in age-related diseases of mammals is largely unknown. Accumulating evidence show that TRPA1 exerts antioxidant and anti-inflammatory effects in various organs and systems (Batai et al., 2019; Kurahara et al., 2018; Takahashi et al., 2018) and that TRPA1 mediates endothelium-dependent cerebral artery dilation (Earley et al., 2009; Pires and Earley, 2018). In addition, TRPA1-induced vasodilatation was reported to be mediated by neuronal NO synthase-derived NO (Aubdool et al., 2016). Interestingly, it was reported that opening TRPA1 consequently phosphorylated protein kinase B and eNOS with concomitant increased NO production in mouse ventricular cardiomyocytes (Andrei et al., 2019). Taken together, TRPA1 has a potential role in the regulation of endothelial function and age-related diseases (Earley, 2012).
Cinnamaldehyde, the major constituent of cinnamon oil, is a selective TRPA1 agonist (Aubdool et al., 2016; Namer et al., 2005). It has been reported that cinnamaldehyde can improve insulin resistance in obese mice and prevents endothelial dysfunction in diabetic mice likely through inhibiting oxidative stress (Camacho et al., 2015; Wang et al., 2015; Wang et al., 2020b). Cinnamaldehyde will be used to investigate the role of TRPA1 in age-related endothelial dysfunction in the present study.
Based on the evidence that TRPA1 is involved in oxidative defense, NO bioavailability regulation, and age-related pathologies, we hypothesized that TRPA1 plays a role in age-related endothelial dysfunction. The present study investigated the role of TRPA1 in replicative senescence of endothelial cells and in endothelial dysfunction of aged mice. This study could provide a potential therapeutic target for the prevention and treatment of age-related endothelial dysfunction and vascular diseases.
Section snippets
Cell culture
Human umbilical vein endothelial cells (HUVECs) were purchased from Schenkel Research Laboratories (San Diego, CA, USA) and were grown in Dulbecco's Modified Eagle's Medium (DMEM, HyClone, Logan, UT, USA). The cells were passaged when they reach 80% confluency. HUVECs were grown from passage 4 to passage 14 which was considered replicative senescence. For intervention experiments, cells were treated with cinnamaldehyde (100 μM, Sigma-Aldrich, St. Louis, MO, USA), cinnamaldehyde plus HC-030031
Activation of TRPA1 reverses senescence-induced eNOS inactivation
HUVECs were grown from the 4th to the 14th passage to induced replicative senescence. The expression levels of TRPA1, eNOS, and p-eNOS in HUVECs were gradually decreased along senescence from the 4th to the 14th passage and were significantly reduced in HUVECs at passage 14 compared with the 4th passage (Fig. 1A–D). Interestingly, treatment with a specific TRPA1 agonist, cinnamaldehyde, reversed senescence-induced downregulation of TRPA1, eNOS, and p-eNOS (Fig. 1A–D). As expected, the rescue
Discussion
The main findings in the present study are that TRPA1 was downregulated in senescent endothelial cells and aged mouse aortas and that activating TRPA1 prevented replicative senescence of endothelial cells and age-related endothelial dysfunction in mice. In addition, the beneficial effects of TRPA1 activation on age-related endothelial dysfunction were associated with reduced oxidative stress and improved NO bioavailability along with phosphorylated eNOS and increased antioxidant proteins Nrf2
Conclusions
In conclusion, the findings in the present study suggest that TRPA1 may be a key player in age-related endothelial dysfunction and may become a therapeutic target for the treatment and prevention of age-related cardiovascular disease.
Ethical statement
All experiments were approved by the Institutional Animal Care and Use Committee of Chengdu Medical College.
Funding
This work was supported by grants from the National Natural Science Foundation of China (81970262, P.W.), Innovation Team Project Education Department of Sichuan Province (18TD0030, P.W.), and Central Funds Guiding the Local Science and Technology Development of Sichuan Province (2020ZYD036, P.W.).
CRediT authorship contribution statement
Yi Yang: Conceptualization, Investigation, Methodology, Data curation, Formal analysis. Dan Wang: Investigation, Methodology, Data curation. Jindong Wan: Investigation, Methodology. Fei Ran: Investigation, Methodology. Lun Yang: Investigation, Methodology. Shizhao Chen: Investigation, Methodology. Fang Wang: Investigation, Methodology. Sen Liu: Investigation, Methodology. Xiaozhen Dai: Investigation, Methodology. Peng Zhou: Supervision. Peijian Wang: Conceptualization, Roles/Writing - original
Declaration of competing interest
Authors declare they do not have any conflict of interest to declare.
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These authors contributed equally to this study.