**3. SIRT1 in endothelial cells: molecular targets and biological functions**

Apart from histones, SIRT1 can mediate the deacetylation of various signaling substrates to exert vasoprotective functions. SIRT1 is abundant in ECs mediating postnatal blood vessel growth via Foxo1 and helps to maintain endothelial function [38]. In vitro experiments showed that downregulation of SIRT1 using small interfering RNA (siRNA) uniquely inhibited endothelial sprout formation via a three-dimensional assay, while other mammalian sirtuin family members (SIRT2–SIRT7) could not [38]. In addition, the reduction of matrix metalloproteinase-14 (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 upregulating SIRT1 expression through the PI3K/Akt/ERK pathway [52].

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 trans-

Targeting Endothelial SIRT1 for the Prevention of Arterial Aging

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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

Limited information is available concerning the role of endothelial SIRT1 in vascular remodeling. In eNOS-deficient mice, overexpression of endothelial SIRT1 prevents hypertension and

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

porter 1 that transport cholesterol into pre-βHDL particles [57].

pathway in ECs.

age-related adverse arterial remodeling [37].
