In a recent study that included data from humans, mice, and cell culture experiments, researchers demonstrated that gut microbes and their metabolites can profoundly influence the senescence of endothelial cells. They also explored the molecular processes underlying these changes [1].
Senescence in the blood vessels
Endothelial cells line the inner surfaces of blood vessels, and their proper functioning is essential for cardiovascular health [2]. However, aging leads to the senescence of endothelial cells and is accompanied by numerous cellular changes, which, in turn, cause endothelial cell dysfunction and contribute to the development of cardiovascular diseases associated with aging [3].
In a recent study, the researchers sought to understand the mechanisms driving endothelial cell senescence. They decided to focus on the potential impact of microbiota-derived metabolites and oxidative stress, as previous research had suggested a possible link between these processes.
The host-microbial connection
The researchers started their study by analyzing plasma metabolites in 24-month-old (aged) and 3-month-old (young) mice. Aged mice had higher plasma concentrations of phenylacetic acid (PAA) and its byproduct, phenylacetylglutamine (PAGln), compared to young mice.
Those two metabolites are interconnected in the metabolic pathway that is shared between the host and microbiome. In this pathway, dietary phenylalanine is converted into PAA by the microbiome, then the host metabolizes PAA into PAGIn.
The researchers observed that both the products and components of this metabolic pathway are elevated in aged mice. Specifically, their analysis of microbial genes indicated that the homologs of two genes essential in converting phenylalanine into PAA, ppfor and vor genes, were more abundant among the microbes in the aged mice, which correlated with an increase in plasma PAA levels. Plasma PAGln levels showed weaker associations.
This pathway plays a crucial role in cardiovascular diseases, as previous research has identified links between PPFOR and atherosclerotic cardiovascular diseases [4]. PAGIn has been associated with heart failure [5] and cellular senescence [6], and PAA has been linked to major adverse cardiovascular events in cardiac patients [7].
Additionally, the researchers identified a bacterium, Clostridium sp. ASF356 MGG37314, which was “the only PPFOR-positive bacterium positively associated with plasma PAA levels in aged mice.” There was also an association between plasma PAGln but not PAA, among five other taxa from the class Clostridia, in aged mice.
To compare whether similar processes occur in humans, they analyzed the data from the TwinsUK cohort. As in murine studies, they observed age-associated increases in PAA and PAGln, as well as enrichment of bacteria belonging to the Clostridium genus harboring the ppfor gene, in older participants of the TwinsUK study. Enrichment in Clostridium correlated more prominently with PAA levels than with PAGIn.
This age-related elevation in the PAA levels and Clostridium was associated with significant endothelial dysfunction, increased cellular senescence biomarkers, and the senescence-associated secretory phenotype (SASP) in the aortic endothelial cells of aged mice. The researchers suggested that the gut microbiome and PAA play key roles in the vascular decline associated with aging.
Aging-promoting processes
To test the effect of Clostridium sp. ASF356 and its metabolite PAA on aortic endothelial senescence and dysfunction, the researchers wiped out the mice’s microbiome with antibiotics and then colonized it with Clostridium sp. ASF356. They observed an increase in both PAA and PAGIn in the Clostridium sp. ASF356-colonized mice.
Analysis of tissues taken from mouse aortic endothelial cells showed multiple signs and markers of endothelial cell senescence and dysfunction. Treating mice with a senolytic cocktail of dasatinib and quercetin reversed major markers of senescence, which correlated with improved vascular function. All of this suggested a direct involvement of Clostridium sp. ASF356 in endothelial senescence in vivo.
To establish the role of PAA in this process, the researchers used a different approach. They treated proliferating human aortic endothelial cells with PAA. This treatment resulted in cellular senescence, as observed by multiple biomarkers. Dasatinib and quercetin treatment of PAA-treated cells reduced the viability of senescent endothelial cells but not proliferating cells. It also reduced some senescence biomarkers and improved the process of new blood vessel formation (angiogenesis).
When PAA was administered to young mice, the researchers observed increased plasma levels of PAA and PAGIn as well as increased levels of senescence markers in aortic cells and worsening markers of vascular health, similar to observations made in young mice colonized with Clostridium sp. ASF356. This suggested “a causal role for PAA in driving endothelial cell senescence.”
Stressing the cells
After establishing a causal link between microbiota and endothelial cell dysfunction, the researchers sought to gain a deeper understanding of this relationship at the molecular level. Previous research has suggested that excessive reactive oxygen species (ROS) trigger endothelial cell senescence [8]. Other studies suggest that gut microbiota regulate oxidative stress-responsive host genes [9].
Combining information from those studies with their results, the researchers investigated whether PAA can increase the levels of H2O2, one of the reactive oxygen species.
When proliferating endothelial cells were exposed to PAA, the researchers observed an increase in ROS levels, specifically mitochondrial H2O2. They also noted that treating proliferating endothelial cells with PAA led to changes in metabolism, and some measures of metabolic activity resembled those seen in senescent endothelial cells.
Further experiments uncovered how the signals are transmitted. They learned that PAA induces mitochondrial H₂O₂ overproduction, leading to increased levels of IL-6, one of the SASP factors. These molecular changes lead to a cascade of further molecular alterations, resulting in the upregulation of the SASP, reduced angiogenesis, epigenetic modifications, energy imbalance, and vascular dysfunction.
Fighting microbes with microbes
Aging changes to the microbiome also include a reduction in the health-promoting short-chain fatty acids (SCFAs), such as acetate, a key anti-inflammatory SCFA, due to the depletion of acetate-producing bacteria. Reduced levels of acetate-producing bacteria are associated with the severity of cardiovascular diseases [10].
Treating PAA-exposed proliferating endothelial cells with sodium acetate exhibited senomorphic effects, including the rescue of proliferation and suppression of the SASP. The researchers also observed reduced levels of senescence markers and DNA damage as well as mitigation of PAA-induced telomere shortening.
Sodium acetate treatment also prevented mitochondrial oxidative stress, improved mitochondrial function, and restored energy balance in PAA-exposed endothelial cells.
On the molecular level, sodium acetate has been previously reported to positively impact nuclear factor erythroid 2-related factor 2 (Nrf2), a molecule regulated by Sirt1 and a master regulator of many antioxidant enzymes [8].
The researchers observed that PAA treatment suppressed Sirt1 and Nrf2, promoting the cytosolic localization of Nrf2 and impairing its antioxidant functions. Acetate treatment restored nuclear Nrf2 expression, which promoted the expression of ROS-neutralizing enzymes.
Sirt1 played a role in this process, as the experiments suggested that acetate-mediated Nrf2 nuclear retention depends on Sirt1. Additionally, the upregulation of Sirt1 by sodium acetate led to the suppression of NF-κB signaling, which, in turn, suppresses the expression of genes encoding SASP components.
Sodium acetate also had a positive impact on PAA-induced angiogenic incompetence. The researchers demonstrated that acetate’s senomorphic properties facilitated the restoration of endothelial function and exerted a pro-angiogenic effect in PAA-induced senescent endothelial cells.
Potential biomarkers and therapeutic approaches
This study broadly suggests that, as the lead author of the study, Seyed Soheil Saeedi Saravi, said, “The aging process of the cardiovascular system can therefore be regulated via the microbiome,” specifically Clostridium sp. ASF356 and its metabolite PAA.
The study suggests a mechanism by which senescence is triggered by PAA-induced mitochondrial H2O2 production, leading to an exacerbated SASP, DNA damage, and proliferative arrest.
The authors suggest using the abundance of Clostridium sp. ASF356 in the microbiome or PAA as potential vascular aging biomarkers.
Beyond explaining the mechanism behind the connection between microbiota and vascular aging, they also identified the role of sodium acetate as a senomorphic agent with pro-angiogenic potential, which could be therapeutically used to promote improvements in endothelial function.
Literature
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