Enalapril mitigates aging by targeting antioxidative genes via phosphorylated Smad1/5/9

Reviewer #1 (Public review):

Summary:

In this study, the authors showed that enalapril was able to reduce cellular senescence and improve health status in aged mice. The authors further showed that phosphorylated Smad1/5/9 was significantly elevated and blocking this pathway attenuated the protection of cells from senescence. When middle-aged mice were treated with enalapril, the physiological performance in several tissues, including memory capacity, renal function, and muscle strength, exhibited significant improvement.

Strengths:

The strength of the study lies in the identification of the pSMAD1/5/9 pathway as the underlying mechanism mediating the anti-senescence effects of enalapril with comprehensive evaluation both in vitro and in vivo.

Weaknesses:

The major weakness of the study is the in vivo data. Despite the evidence shown in the in vitro study, there is no data to show that blocking the pSmad1/5/9 pathway is able to attenuate the anti-aging effects of enalapril in the mice. In addition, the aging phenotypes mitigation by enalapril is not evidenced by the extension of lifespan. If it is necessary to show that NAC is able to attenuate enalapril effects in the aging mice. In addition, it would be beneficial to test if enalapril is able to achieve similar rescue in a premature aging mouse model.

Reviewer #2 (Public review):

This manuscript presents an interesting study of enalapril for its potential impact on senescence through the activation of Smad1/5/9 signaling with a focus on antioxidative gene expression. Repurposing enalapril in this context provides a fresh perspective on its effects beyond blood pressure regulation. The authors make a strong case for the importance of Smad1/5/9 in this process, and the inclusion of both in vitro and in vivo models adds value to the findings. Below, I have a few comments and suggestions which may help improve the manuscript.

A major finding in the study is that phosphorylated Smad1/5/9 mediates the effects of enalapril. However, the manuscript focused on the Smad pathway relatively abruptly, and the rationale behind targeting this specific pathway is not fully explained. What makes Smad1/5/9 particularly relevant to the context of this study?

Furthermore, their finding that activation of Smad1/5/9 leads to a reduction of senescence appears somewhat contradictory to the established literature on Smad1/5/9 in senescence. For instance, studies have shown that BMP4-induced senescence involves the activation of Smad1/5/8 (Smad1/5/9), leading to the upregulation of senescence markers like p16 and p21 (JBC, 2009, 284, 12153). Similarly, phosphorylated Smad1/5/8 has been shown to promote and maintain senescence in Ras-activated cells (PLOS Genetics, 2011, 7, e1002359). Could the authors provide more detailed mechanistic insights into why enalapril seems to reverse the typical pro-senescent role of Smad1/5/9 in their study?

While the authors showed that enalapril increases pSmad1/5/9 phosphorylation, what are the expression levels of other key and related factors like Smad4, pSmad2, pSmad3, BMP2, and BMP4 in both senescent and non-senescent cells? These data will help clarify the broader signaling effects.

They used BMP receptor inhibitor LDN193189 to pharmacologically inhibit BMP signaling, but it would be more convincing to also include genetic validation (e.g., knockdown or knockout of BMP2 or BMP4). This will help confirm that the observed effects are truly due to BMP-Smad signaling and not off-target effects of the pharmacological inhibitor LDN.

I don't see the results on the changes in senescence markers p16 and p21 in the mouse models treated with enalapril. Similarly, the effects of enalapril treatment on some key SASP factors, such as TNF-α, MCP-1, IL-1β, and IL-1α, are missing, particularly in serum and tissues. These are important data to evaluate the effect of enalapril on senescence.

Given that enalapril is primarily known as an antihypertensive, it would be helpful to include data on how it affects blood pressure in the aged mouse models, such as systolic and diastolic blood pressure. This will clarify whether the observed effects are independent of or influenced by changes in blood pressure.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this study, the authors showed that enalapril was able to reduce cellular senescence and improve health status in aged mice. The authors further showed that phosphorylated Smad1/5/9 was significantly elevated and blocking this pathway attenuated the protection of cells from senescence. When middle-aged mice were treated with enalapril, the physiological performance in several tissues, including memory capacity, renal function, and muscle strength, exhibited significant improvement.

Strengths:

The strength of the study lies in the identification of the pSMAD1/5/9 pathway as the underlying mechanism mediating the anti-senescence effects of enalapril with comprehensive evaluation both in vitro and in vivo.

Thanks very much for your insightful evaluation and the constructive suggestions. We have thoroughly studied the comments and a provisional point-to-point response is shown as follows.

Weaknesses:

The major weakness of the study is the in vivo data. Despite the evidence shown in the in vitro study, there is no data to show that blocking the pSmad1/5/9 pathway is able to attenuate the anti-aging effects of enalapril in the mice. In addition, the aging phenotypes mitigation by enalapril is not evidenced by the extension of lifespan.

Thanks for your comment. As suggested, we will feed LDN193189 to mice while using LDN193189 to block pSmad1/5/9, and will assess age-related phenotypes in the mice to demonstrate that the anti-aging effect of enalapril in mice is mediated through pSmad1/5/9.

We only assess the improvement in the health status of the aging mice, which indicate that enalapril can extend the healthy lifespan of aging mice. This is because we believe that lifespan is controlled by genetics. Therefore, this study focuses solely on the improvement of health phenotypes in aging mice by enalapril.

If it is necessary to show that NAC is able to attenuate enalapril effects in the aging mice. In addition, it would be beneficial to test if enalapril is able to achieve similar rescue in a premature aging mouse model.

Thanks for your suggestion. To our knowledge, NAC is an inhibitor of ROS, which is consistent with the antioxidant effect of enalapril. Therefore, we believe that NAC will not diminish the effect of enalapril.

For the premature aging mouse models, we examined the effect of enalapril on Lmna^G609G mice and other premature aging models and found that the effect was relatively modest. This may be due to differences in the genetic background of premature aging mice, leading to a less pronounced effect of enalapril compared to its impact on naturally aged mice.

Reviewer #2 (Public review):

This manuscript presents an interesting study of enalapril for its potential impact on senescence through the activation of Smad1/5/9 signaling with a focus on antioxidative gene expression. Repurposing enalapril in this context provides a fresh perspective on its effects beyond blood pressure regulation. The authors make a strong case for the importance of Smad1/5/9 in this process, and the inclusion of both in vitro and in vivo models adds value to the findings. Below, I have a few comments and suggestions which may help improve the manuscript.

Thanks very much for your insightful evaluation and the constructive suggestions. We have thoroughly studied the comments and a provisional point-to-point response is shown as follows.

A major finding in the study is that phosphorylated Smad1/5/9 mediates the effects of enalapril. However, the manuscript focused on the Smad pathway relatively abruptly, and the rationale behind targeting this specific pathway is not fully explained. What makes Smad1/5/9 particularly relevant to the context of this study?

Thanks for your comment. As stated in the manuscript, after we found that enalapril could improve the cellular senescence phenotype, we screened and examined key targets in important aging-related signaling pathways, such as AKT, mTOR, ERK (Fig. S2A), Smad2/3 and Smad1/5/9 (Fig. 2A). We found that only the phosphorylation levels of Smad1/5/9 significantly increased after enalapril treatment. Therefore, the subsequent focus of this study is on pSmad1/5/9.

Furthermore, their finding that activation of Smad1/5/9 leads to a reduction of senescence appears somewhat contradictory to the established literature on Smad1/5/9 in senescence. For instance, studies have shown that BMP4-induced senescence involves the activation of Smad1/5/8 (Smad1/5/9), leading to the upregulation of senescence markers like p16 and p21 (JBC, 2009, 284, 12153). Similarly, phosphorylated Smad1/5/8 has been shown to promote and maintain senescence in Ras-activated cells (PLOS Genetics, 2011, 7, e1002359). Could the authors provide more detailed mechanistic insights into why enalapril seems to reverse the typical pro-senescent role of Smad1/5/9 in their study?

Thanks for your comment. The downstream regulatory network of BMP-pSmad1/5/9 is highly complex. The BMP-SMAD-ID axis has been mentioned in many studies, and its downstream signaling inhibits the expression of p16 and p21 (PNAS, 2016, 113(46), 13057-13062; Cell, 2003, 115(3), 281-292). Additionally, studies have also found that the Smad1-Stat1-P21 axis inhibits osteoblast senescence (Cell Death Discovery, 2022, 8:254). In our study, enalapril was found to increase the expression of ID1, which is a classic downstream target of pSmad1/5/9 (Cell Stem Cell, 2014, 15(5), 619-633). Therefore, pSmad1/5/9 inhibits cellular senescence markers such as p16, p21 and SASP through ID1, thereby promoting cell proliferation (Fig. 3). Furthermore, we also found that pSmad1/5/9 increases the expression of antioxidant genes and reduces ROS levels, exerting antioxidant effects (Fig. 4). Together, ID1 and antioxidant genes enable pSmad1/5/9 to exert its anti-aging effects.

While the authors showed that enalapril increases pSmad1/5/9 phosphorylation, what are the expression levels of other key and related factors like Smad4, pSmad2, pSmad3, BMP2, and BMP4 in both senescent and non-senescent cells? These data will help clarify the broader signaling effects.

Thanks for your suggestion. We observed an increase in Smad4 expression, while the levels of pSmad2 and pSmad3 remained unchanged after enalapril treatment (Fig. 2A). We will supplement data on the expression changes of these key factors in both senescent and non-senescent cells.

They used BMP receptor inhibitor LDN193189 to pharmacologically inhibit BMP signaling, but it would be more convincing to also include genetic validation (e.g., knockdown or knockout of BMP2 or BMP4). This will help confirm that the observed effects are truly due to BMP-Smad signaling and not off-target effects of the pharmacological inhibitor LDN.

Thanks for your suggestion. We will use shRNA or siRNA to knockdown BMP and examine the related changes to clarify the role of BMP-Smad signaling.

I don't see the results on the changes in senescence markers p16 and p21 in the mouse models treated with enalapril. Similarly, the effects of enalapril treatment on some key SASP factors, such as TNF-α, MCP-1, IL-1β, and IL-1α, are missing, particularly in serum and tissues. These are important data to evaluate the effect of enalapril on senescence.

Thanks for your comment. As for the markers p16 and p21, we observed no change in p16, while the changes in p21 varied across different organs and tissues. (Author response image 1). Nevertheless, behavioral experiments and physiological and biochemical indicators at the individual level consistently demonstrated the significant anti-aging effects of enalapril (Fig. 6).

Author response image 1.

p21(Cdkn1a) expression levels in organs of mice after enalapril feeding.

We also examined the changes in SASP factors in the serum of mice after enalapril treatment. Notably, SASP factors such as CCL (MCP), CXCL and TNFRS11B showed significant decreases (Fig. 5C). The expression changes of SASP factors varied across different organs. In the liver, kidneys and spleen, the expression of IL1a and IL1b decreased, while TNFRS11B expression decreased in both the liver and muscles (Fig. 5B). Additionally, CCL (MCP) levels decreased in all organs (Fig. 5B).

Given that enalapril is primarily known as an antihypertensive, it would be helpful to include data on how it affects blood pressure in the aged mouse models, such as systolic and diastolic blood pressure. This will clarify whether the observed effects are independent of or influenced by changes in blood pressure.

Thanks for your comment. We measured the blood pressure in mice, and found no significant change in blood pressure after enalapril treatment, which has also been validated in other studies (J Gerontol A Biol Sci Med Sci, 2019, 74(8), 1149–1157). Therefore, our results are independent of changes in blood pressure.