**4. Endothelial SIRT1 prevents arterial aging**

Various animal studies demonstrated that SIRT1 plays a vital role in anti-endothelial senescence and anti-atherogenesis. Infiltration of monocyte-derived macrophages into the subendothelial space is a crucial step in atherogenesis [53]. SIRT1 can decrease cholesterol uptake especially oxLDL and prevent macrophage foam cell formation via suppressing the expression of scavenger receptor Lox-1 [54] and reducing the expression of various pro-inflammatory molecules including TNF-α, monocyte chemotactic protein-1, and interleukins [55]. A recent discovery showed that treatment of the SIRT1 activator SRT3025 decreased plasma levels of LDL cholesterol and total cholesterol and attenuated atherosclerosis, owing to reduced secretion of hepatic Pcsk9 and enhanced protein expression of LDL receptor in apolipoprotein E-deficient (ApoE−/−) mice [56]. In the meantime, SIRT1 was demonstrated to promote reverse cholesterol (mainly HDLs) transport into macrophages by directly deacetylating and subsequently regulating the transcriptional activity of liver X receptors, which play a significant role in lipid homeostasis and inflammation and can help express ATP-binding cassette transporter 1 that transport cholesterol into pre-βHDL particles [57].

(MMP-14), a membrane-anchored MMP essential for tip cell activity during sprouting angiogenesis, was found in siRNA-SIRT1-treated endothelial sprouts [39]. Decreased expression of SIRT1 either by mRNA silencing or pharmacological inhibition could induce prematuresenescence-like phenotypes in ECs [40, 41]. SIRT1 displays anti-senescence activity in ECs by inducing the deacetylation of diversified signaling substrates [42]. For example, SIRT1 can deacetylate tumor suppressor protein p53 to downregulate its stability and activity as to promote cell survival in response to cellular stress [43]. SIRT1 also plays an important role in enhancing the endothelial NO synthase (eNOS) transcription and translation by deacetylating eNOS on lysine 496 and 506 to generate more NO, thus enhancing vessel dilatation, mediating vessel tone regulation, and providing athero-protective effects [44, 45]. Recent study demonstrated that SIRT1 activation could help reduce traction forces and reorganize actin localization (increased peripheral actin) in aged ECs, which is also a sign of anti-senescent effect [46]. Moreover, while senescent porcine aortic ECs (PAECs) showed decreased expression of SIRT1 compared to young PAECs, the protein level of liver kinase B1 (LKB1), a serine/threonine kinase and tumor suppressor, was dramatically increased as well as the phosphorylation of its downstream target AMPK (Thr172). In this case, SIRT1 can antagonize LKB1-dependent AMPK activation by promoting the deacetylation, ubiquitination, and proteasome-mediated degradation in order to retard PAEC senescence which also correlated with the Akt survival signaling pathway [41]. Furthermore, it was reported that SIRT1 can bind to the DOC domain of HERC2 [HECT and RLD domain containing E3 ubiquitin protein ligase 2] and then ubiquitinate LKB1 in the nuclear compartment of ECs [37]. SIRT1 can also negatively modulate Notch signaling in ECs via deacetylation of the Notch1 intracellular domain (NICD), in which loss of endothelial SIRT1 activity leads to impaired growth and sprout elongation [47]. Intracellular NAMPT-NAD+-SIRT1 cascade was shown to improve post-ischemic neovascularization through modulation of Notch signaling pathway [48]. Adapter protein p66Shc which can directly stimulate mitochondrial reactive oxygen species (ROS) generation was discovered downregulated by SIRT1 in mice with hyperglycemia-induced endothelial dysfunction [49]. Moreover, in vitro experiments using human aortic ECs (HUVECs) demonstrated that SIRT1 can deacetylate RelA/p65 to diminish tissue factor expression and suppress nuclear factor-κB (NF-κB) signaling, thus preventing atherothrombosis [50]. In EPCs, SIRT1 was implicated to protect against oxidative stress-induced apoptosis by inhibiting Foxo3a via ubiquitination and degradation [36]. microRNA-34a (miR-34a), regulated by p53 and able to control cell cycle arrest, has been reported to promote cardiac, endothelial, and EPC senescence via downregulation of SIRT1 [51]. Also, visfatin (an adipocytokine closely associated with human cell senescence) was reported to attenuate the oxLDL-induced senescence of EPCs by upregulat-

78 Endothelial Dysfunction - Old Concepts and New Challenges

ing SIRT1 expression through the PI3K/Akt/ERK pathway [52].

Various animal studies demonstrated that SIRT1 plays a vital role in anti-endothelial senescence and anti-atherogenesis. Infiltration of monocyte-derived macrophages into the subendothelial space is a crucial step in atherogenesis [53]. SIRT1 can decrease cholesterol uptake especially oxLDL and prevent macrophage foam cell formation via suppressing the expression

**4. Endothelial SIRT1 prevents arterial aging**

Some studies regarding upstream regulators of SIRT1 including cathepsin, caspase-1, and cyclin-dependent kinase 5 (CDK5) elucidate beneficial roles of SIRT1 in anti-endothelial senescence and anti-atherogenesis [58–60]. The cysteine cathepsins belong to the leaked lysosomal contents with the viability in cleavage and degradation of SIRT1, which lead to stressinduced premature senescence [58]. Studies on ApoE−/−/caspase-1−/− double knockout mice have shown promising evidences that early hyperlipidemia promoted endothelial activation via a Caspase-1-SIRT1 pathway [59]. In this case, researchers found that inhibition of caspase-1 resulted in SIRT1 accumulation in the ApoE−/− mouse aorta and ApoE−/−/caspase-1−/− mice had attenuated early atherosclerosis, decreased aortic expression of proinflammatory cytokines, and reduced aortic monocyte recruitment, as well as decreased endothelial activation [59]. Another upstream regulator of SIRT1 is CDK5, which was proved to increase the phosphorylation of SIRT1 especially at S47 during cellular senescence [60]. In this study, replacing S47 with nonphosphorable alanin (S47A) elevated, while mutation of S47 to phospho-mimicking aspartic acid (S47D) abolished the beneficial effects of SIRT1 such as anti-senescence, growth promotion, and downregulation of LKB1 expression [60]. Interaction between SIRT1 and telomeric repeat-binding factor 2-interacting protein 1 was abolished when S47 was phosphorylated. NF-κB signaling pathway is activated to induce endothelial inflammation and leads to endothelial senescence and atherosclerosis. Downregulation of CDK5 by either knockdown (by siRNA) or inhibition (by roscovitine) reduced percentage of senescent ECs and attenuated inflammatory gene expression. Meanwhile, long-term treatment of ApoE−/− mice with the CDK5 inhibitor, roscovitine, resulted in attenuated atherosclerosis in aortae [60]. As CDK5R1(p35/p25) is the crucial activator mediating the kinase activity of CDK5 [60, 61], further research will be conducted to unveil the underlying mechanism of CDK5-p35/p25-SIRT1 pathway in ECs.

Limited information is available concerning the role of endothelial SIRT1 in vascular remodeling. In eNOS-deficient mice, overexpression of endothelial SIRT1 prevents hypertension and age-related adverse arterial remodeling [37].

Laminar shear stress is an important stimulus for the endothelium-dependent control of vascular tone and of vascular remodeling processes. In cultured ECs, laminar flow increases both the expression and activity of SIRT1, whereas oscillating flow decreases SIRT1 expression [62]. In mouse arteries, the formation of neointima is accompanied by a progressive downregulation of SIRT1 expression [63]. SIRT1 inhibition in ECs increases the expressions of p53 and its downstream target, plasminogen activator inhibitor-1 (PAI-1), which promotes the formation of neointima and vascular remodeling in response to vascular injury [40].

apoptosis, and oxidative stress [72]. It was found to dramatically lower the incidence of cardiovascular diseases in spite of high saturated fat diet, which was termed as "French paradox" [73]. Several natural ingredients extracted from various traditional Chinese herbs were also found to activate SIRT1. Tetramethylpyrazine is proved to reverse high-glucoseinduced endothelial dysfunction via SIRT1 [74]. Polydatin can attenuate hemorrhagic shock by upregulating SIRT1 [75]. Quercetin is capable of inhibiting oxidized LDL-induced EC damage by SIRT1 activation [76]. Some other natural polyphenols including fisetin and butein can also activate SIRT1 [77]. Vitamin D protects ECs from irradiation-induced senescence and apoptosis by modulating MAPK/SirT1 axis [78]. On the other hand, a series of SIRT1 activators like SRT2183, SRT1460, SRT1720, SRT2379, SRT501, SRT2104, SRT3025, and BMT0-512 have been synthesized and developed as potential drugs to protect against vascular aging [77, 79]. Despite the fact that SIRT1 is as an optimal therapeutical target for cardiovascular diseases, the dosage of upregulation of SIRT1 should be considered seriously and titrated cautiously in clinical practice. It was reported that 2.5- to 7-fold overexpression of SIRT1 prevented heart from oxidative stress via SIRT1/FOXO, while 12.5-fold overexpression of SIRT1 increased apoptosis and hypertrophy and decreased cardiac function, suggesting that only low to moderate doses of SIRT1 can exert beneficial effects [80]. The aforementioned findings call for more careful evaluation of dosage and possible adverse effects in drug development targeting

Targeting Endothelial SIRT1 for the Prevention of Arterial Aging

http://dx.doi.org/10.5772/intechopen.73019

81

In light of all the above studies, there have already been several potential drugs to target the anti-vascular aging effects of endothelial SIRT1. The entry of SIRT1 activators into human trials is exciting but also highlights the necessity to better understand the SIRT1 specificity,

This work was financially supported by Hong Kong Health and Medical Research Fund

Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong

clinical effects, and side effects of these promising activators in vivo.

endothelial SIRT1.

**Acknowledgements**

**Conflict of interest**

**Author details**

Yumeng Guo and Yu Wang\*

No interest of conflict was declared.

\*Address all correspondence to: yuwanghk@hku.hk

13142651.

Loss of vascular smooth muscle cell (VSMC) function is an alarming sign of vascular disease. During the aging process, VSMCs undergo increased dysregulation, apoptosis, and senescence [64]. In VSMCs, SIRT1 can act as a modulator of neointima formation (associated with repression of activator protein-1 (AP-1) activity [63]) and protect against DNA damage. Aging-related loss of SIRT1 expression correlates with lower capacity for vascular repair, abolished stress response, and elevated senescence [63].

Decreased expression of SIRT1 in VSMCs exerts its proatherogenic effects by the failure to deacetylate histones in DNA repair, response to oxidant stress and LDL, and therefore leads to VSMC senescence and apoptosis [63, 65]. As to atherosclerotic plaques, SIRT1 activity has been suggested to deacetylate the regulatory factor for X-box (RFX5) and antagonized repression of collagen type I (COL1A2) transcription in VSMCs, consequently stabilizing the plaque and avoiding rupture [66]. Another most recent finding relevant to destabilization of atherosclerotic plaque is that SIRT1 participated in downregulation of platelet-activating factor receptor (PAFR) in VSMCs through β-arrestin 2-mediated internalization and degradation, resulting in the inhibition of PAF-induced matrix metalloproteinase (MMP-2) generation [67]. In addition, inhibition of miR-138 was found to increase SIRT1 expression in VSMCs separated from diabetic (db/db) mice and in SMC lines C-12511 in recent study, which indicated miR-138 as another potential inhibitory target to attenuate the proliferation and migration of VSMCs and cure atherosclerosis [68]. Furthermore, SIRT1 was also found to inhibit angiotensin II-induced VSMC hypertrophy in rat embryonic aortic VSMCs [69]. Later on, SIRT1 demonstrated antihypertensive activity in transgenic mice with selective overexpression of SIRT1 in VSMCs (SV-Tg). Alleviated vascular remodeling in mouse thoracic and renal aortae induced by angiotensin II is observed, along with significantly decreased transforming growth factor-β1 (TGF-β1) expression, ROS generation, vascular inflammation, and collagen formation in the arterial wall of SV-Tg mice [70]. Similar to contribution in ECs, overexpression of miR-34a can upregulate p21 level and inflammation through SIRT1 downregulation and cause senescence-associated secretory phenotype factors induction (including proinflammatory molecules such as cytokines, chemokines, proteases, growth factors, soluble receptors, etc.), promoting VSMC senescence and leading to arterial dysfunction [71].
