Activation of DNA demethylases attenuates aging‐associated arterial stiffening and hypertension

Summary DNA methylation increases with age. The objective of this study was to investigate whether compound H, a potential activator of DNA demethylases, attenuates aging‐related arterial stiffness and hypertension. Aged mice (24–27 months) and adult mice (12 months) were used. Pulse wave velocity (PWV), a direct measure of arterial stiffness, and blood pressure (BP) were increased significantly in aged mice. Notably, daily treatments with compound H (15 mg/kg, IP) for 2 weeks significantly attenuated the aging‐related increases in PWV and BP. Compound H abolished aging‐associated downregulation of secreted Klotho (SKL) levels in both kidneys and serum likely by enhancing DNA demethylase activity and decreasing DNA methylation. Aging‐related arterial stiffness was associated with accumulation of stiffer collagen and degradation of compliant elastin which are accompanied by increased expression of MMP2, MMP9, TGF‐β1, and TGF‐β3. These changes were effectively attenuated by compound H, suggesting rejuvenation of aged arteries. Compound H also rescued downregulation of Sirt1 deacetylase, AMPKα, and eNOS activities in aortas of aged mice. In cultured smooth muscle cells (SMCc) Klotho‐deficient serum upregulated expression of MMPs and TGFβ which, however, was not affected by compound H. In conclusion, compound H attenuates aging‐associated arterial stiffness and hypertension by activation of DNA demethylase which increases renal SKL expression and consequently circulating SKL levels leading to activation of the Sirt1‐AMPK‐eNOS pathway in aortas of aged mice.

methylation led to arterial stiffening has never been determined. Physiologically, an appropriate methylation level is maintained by the balanced methyltransferase and demethylase activity (Wagner et al., 2016). In this study, we assessed if activation of the demethylase affects arterial stiffening and hypertension in aged mice.
The Klotho gene was originally identified as a putative aging-suppressor gene in mice that extended lifespan when overexpressed and caused multiple premature aging phenotypes when disrupted (Kuro-o et al., 1997;Kurosu et al., 2005). The Klotho level decreases with age (Xiao, Zhang, Zheng & Gu, 2004), while the prevalence of arterial stiffness and hypertension increases with age (Kotsis & Stabouli, 2011). At age 70 years, the serum level of Klotho is only about one half of what it was at age 40 years (Xiao et al., 2004). Moreover, the serum Klotho level is significantly decreased in patients with arterial stiffness in chronic kidney diseases (Karras et al., 2012). Our recent study showed that haplodeficiency of Klotho gene caused arterial stiffness (Chen, Zhou & Sun, 2015). We found, in the cultured renal tubule cells, that a small compound (compound H) may be a potential inducer of Klotho gene expression (Jung, Xu & Sun, 2017). Whether compound H promotes Klotho expression and release in vivo has never been determined. In this study, we investigated whether compound H increases Klotho levels and attenuates aging-associated arterial stiffening and hypertension.
Our results demonstrated that aging-related arterial stiffening and hypertension are attributed, at least in part, to the increased DNA methylation. Compound H activates demethylases and attenuates arterial stiffening and hypertension in aged mice likely via increasing the Klotho levels.

| ME TH ODS
A detailed method section is available in the Data S1.

| Animal study protocols
Six adult mice (12 months) and 20 old mice (24-27 months) were used in this study (129/sv). The old mice were randomly divided into three subgroups and each group had equal number of male mice. Three doses of compound H were tested, and 10 mg/kg bw was chosen as an optimal dose for treating animals. No obvious toxic effect of compound H was observed at this dose. One subgroup received compound H (10 mg kg À1 day À1 , IP, Enamine LLC, Monmouth Jct., NJ) and one group received an equal volume of dimethyl sulfoxide (5%) and served as a control. The structure of compound H was provided in Figure S1. The third group received no treatment. Blood pressure (BP) was measured before and after treatment with compound H at 1 and 2 weeks. Pulse wave velocity (PWV) was measured after 2 weeks treatment with compound H. All animals were euthanized (ketamine/xylazine, 90/10 mg, IP) and perfused transcardially with PBS. The aortas were then quickly removed, washed, and cut into pieces for subsequent analyses.

| Histological and immunohistochemical (IHC) examination
The histological and IHC analysis was performed as described in our recent studies Gao et al., 2016;Lin & Sun, 2015a,b;Varshney, Ali, Wu & Sun, 2016). For details, see Data S1.

| Western blot analysis
For details, see Data S1.

| Methylation analyses of Klotho gene
For details, see Data S1.
2.6 | Measurement of DNA demethylase and DNA methyltransferase activity For details, see Data S1.

| Measurement of MMPs activity
For details, see Data S1.

| Cell culture and treatment
For details, see Data S1.

| Statistical analysis
Quantitative data were presented as the means AE SE. Differences between experimental groups were examined by one-way analysis of variance (ANOVA) followed by the Bonferroni post-test using Prism software (GraphPad, La Jolla, CA). For all analysis, p < .05 were considered statistically significant.

| Compound H attenuated arterial stiffness and hypertension in aged mice
Pulse wave velocity, a direct measure of arterial stiffness, was increased significantly in aged mice (Figure 1a). The widening of pulse pressure is another direct surrogate of arterial stiffness. Pulse pressure of aged mice was significantly increased compared to that of adult mice ( Figure 1b). Thus, aged mice developed arterial stiffness. Daily treatment with compound H for 2 weeks remarkably decreased PWV and pulse pressure in aged mice (Figure 1a

| Compound H increased secreted Klotho (SKL) expression in aged mice
To investigate the potential mechanism contributing to the beneficial effects of compound H on aging-related arterial stiffening, we measured Klotho expression levels in kidneys, a major source of circulating Klotho (Lindberg et al., 2014;Xu & Sun, 2015b). We found that

| Compound H attenuated DNA hypermethylation of Klotho gene in aged mice
To further explore the potential mechanism of induction of Klotho expression by compound H, we measured the DNA demethylase activity and DNA methyltransferase activity in kidneys. The DNA demethylase activity was decreased while the DNA methyltransferase activity was increased in aged mice (24-27 months old) in relative to those of adult mice (12 months old) (Figure 3a,b).
Compound H rescued the downregulation of DNA demethylase activity but did not affect DNA methyltransferase activity in aged mice (Figure 3a,b). We then measured methylation of the Klotho gene. The methylation of CpG islands of the Klotho gene was significantly increased in aged mice compared to that of adult mice (

| DISCUSSION
SKL may function as a hormone (Xu & Sun, 2015). The circulating SKL may have direct action in vascular endothelial cells or SMCs which do not express SKL. Klotho is an aging-suppressor gene (Xu & Sun, 2015). Klotho gene deficiency led to arterial stiffening . This study provides the first evidence that an increase in serum SKL attenuated arterial stiffening. Aging is associated with increased cardiovascular complications, such as arterial stiffness and hypertension (Sun, 2015). Arterial stiffness is an independent risk factor for myocardial infarction, cognitive decline in aging, stroke, and kidney diseases (Boutouyrie et al., 2002;Matsuoka et al., 2005;Waldstein et al., 2008;Zoungas & Asmar, 2007). Unfortunately, the current antihypertensive drugs sion. In a recent study, we showed that compound H directly stimulates DNA demethylase activity, decreases DNA methylation, and enhances Klotho expression in the cultured kidney cells (Jung, Xu & Sun, 2017). Our recent study showed that haplodeficiency of Klotho gene caused arterial stiffness and hypertension .

Adult Old Con DMSO ComH Methylation Unmethylation Input Control
Therefore, we believe that the mechanism of the antihypertensive effect of compound H includes enhanced circulating Klotho levels.  (Xiao et al., 2004), while the prevalence of arterial stiffness and hypertension is increased in the aged population (Kotsis & Stabouli, 2011 Figure S3). This hypothesis needs to be validated.
It is noticed that compound H reversed aging-associated arterial stiffening and remodeling, suggesting that compound H via induction of SKL expression and release rejuvenates aged arteries. The beneficial effect of compound H may be partially attributed to reversal of upregulation of collagen and downregulation of elastin ( Figure 4).
This process may include collagen degradation and elastin synthesis which may be likely due to upregulation of MMP and TGFb induced by increased SKL levels. Although aging led to a decrease in autophagy, the beneficial effect of compound H may not involve autophagy which was not affected by compound H ( Figure S4).
Two types of Klotho protein with potentially different functions have been identified: a full-length transmembrane Klotho and a SKL (Xu & Sun, 2015). The full-length Klotho is mainly expressed in kidney distal tubule cells and serves as a coreceptor of FGF23 and enhances FGF23 signaling to maintain phosphate homeostasis. The circulating Klotho, that is SKL, may act as a hormone and regulate the functions of tissues or cells that do not express Klotho (e.g., vascular endothelial cells and smooth muscle cells) (Lindberg et al., 2013;Xu & Sun, 2015b). In this study, we found that both full-length and SKL in kidneys were decreased in aged mice ( Figure 2). However, compound H only rescued the downregulation of SKL but did not affect transmembrane Klotho. The molecular mechanism of the selective action of compound H on SKL remains to be explored. SKL and the full-length klotho are encoded by the same gene. SKL was generated due to alternative splicing (Xu & Sun, 2015). Thus, compound H might demethylase an alternative RNA splicer to rescue the aging-related downregulation of SKL. This hypothesis warrants further investigation. SIRT1, known as a class III histone deacetylase, is a nuclear protein implicated in the regulation of many cellular processes, including apoptosis, cellular senescence, endocrine signaling, glucose homeostasis, aging, and longevity. SIRT1 has been reported to deacetylate the lysine residues of a number of nuclear proteins, such as p53 (Yuan et al., 2011), NF-jB (Salminen & Kaarniranta, 2009), PGC-1a (Amat et al., 2009), CBP/p300 (Das, Lucia, Hansen & Tyler, 2009), and forkhead family proteins (Brunet et al., 2004). Several recent studies demonstrated that Sirt1 could inhibit TGF-b signaling and ameliorate fibrosis (Huang et al., 2014;Kume et al., 2007;Zerr et al., 2014). In this study, we found that aging suppressed SIRT1 activity, increased TGFb and MMP expressions, and induced fibrosis. Compound H rescued aging-associated downregulation of SIRT1 expression in aortas likely by increasing the circulating SKL levels.
Therefore, downregulation of SIRT1 activity may be an important factor contributing to aging-induced arterial remodeling and stiffness.
However, the recent studies showed that large-artery stiffness precedes the development of hypertension (Kaess et al., 2012;Najjar et al., 2008). Although hypertension could also contribute to vascular remodeling and stiffening, it is, however, a slow process. A reduction in BP by 20 mmHg may be insufficient to reverse aging-related arterial remodeling or arterial stiffening in 2 weeks. Nevertheless, this study does not exclude the possibility that attenuation of BP by compound H may also contribute to the improvement of arterial stiffening in aging mice. The limitation of this study is that it does not elucidate the relationship of arterial stiffening and hypertension (causality).
It is noticed that compound H decreased aging-associated arterial stiffening and hypertension within 2 weeks, which is sooner than expected. This finding suggests that there may be a functional component in arterial stiffening, that is, increased vascular tone. The acute effect of compound H may be partially due to improved endothelial function and decreased vascular resistance.
Indeed, treatment with compound H, via stimulation of SKL release, activated the SIRT1-AMPKa-eNOS pathway as evidenced by increased activities of SIRT1, AMPKa, and eNOS . eNOS is an important enzyme in the regulation of BP, vascular tone, and angiogenesis. Several protein kinases including AMPK activate eNOS by phosphorylating Ser1177 in response to various stimuli (Dimmeler et al., 1999;Fulton et al., 1999). Therefore, the mechanism of the antihypertensive effect of compound H may include activation of the SIRT1-AMPK-eNOS pathway, inhibition of MMP and TGFb, and attenuation of arterial remodeling and stiffening.

| PERSPECTIVE
Our study provides the first experimental evidence that aging-associated arterial stiffening and hypertension are attributed, at least in part, to increased DNA methylation. It is new and interesting that compound H activates DNA demethylase which induces Klotho expression and release leading to attenuation of arterial stiffening and hypertension in aged mice. Our study also provides the first evidence that compound H may be an effective therapeutic agent for aging-related arterial stiffening and hypertension.

CONF LICT OF I NTEREST
None declared.

AUTHORS' CONTRI BUTION
ZS, design experiment, critical editing manuscript; KC, perform experiment, collect data, and write manuscript.