Impairment of an endothelial NAD(+)-H2S signaling network is a reversible cause of vascular aging

“A major cause of age-associated endothelial dysfunction is an increase in oxidative stress and apoptosis (Harman, 1956).”

“Increasing SIRT1 activity has been thought to improve endurance capacity by increasing mitochondrial function (Bai et al., 2011a, 2011b; Baur et al., 2006; Cantó et al., 2009; Lagouge et al., 2006; Pearson et al., 2008).”

“Increasing SIRT1 activity has been thought to improve endurance capacity by increasing mitochondrial function (Bai et al., 2011a, 2011b; Baur et al., 2006; Cantó et al., 2009; Lagouge et al., 2006; Pearson et al., 2008).”

“Increasing SIRT1 activity has been thought to improve endurance capacity by increasing mitochondrial function (Bai et al., 2011a, 2011b; Baur et al., 2006; Cantó et al., 2009; Lagouge et al., 2006; Pearson et al., 2008).”

“Differences in exercise capacity are commonly attributable to changes in muscle fiber type (Pette and Staron, 2000) or mitochondrial content (Lin et al., 2002).”

“SIRT1 is amember of the sirtuin family of NAD-dependent deacylases that mediate the health benefits of dietary restriction (DR) and can extend lifespan when overexpressed (Guarente, 2013; Haigis and Sinclair, 2010; Kanfi et al., 2012; Satoh et al., 2013).”

“A more recent approach has been to restore NAD levels by treating with NAD precursors such as nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN) (Cantó et al., 2015; Gomes et al., 2013; Mouchiroud et al., 2013; Ramsey et al., 2008).”

“SIRT1-iKO mice were fed tamoxifen (360 mg/kg) diet for five weeks to elicit whole-body SIRT1 exon 4 excision as described previously (Price et al., 2012).”

“A more recent approach has been to restore NAD+ levels by treating with NAD precursors such as nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN) (Cantó et al., 2015; Gomes et al., 2013; Mouchiroud et al., 2013; Ramsey et al., 2008).”

“, 2012), and protect against age-related physiological decline including reduced DNA repair, mitochondrial dysfunction, and glucose intolerance (de Picciotto et al., 2016; Gomes et al., 2013; Li et al., 2017; Yoshino et al., 2011).”

“Mice treated with NMN or NR for extended periods show no evidence of increased tumor burden (Mills et al., 2016; Zhang et al., 2016).”

“, 2014), improve the exercise capacity of young mice (Cantó et al., 2012), and protect against age-related physiological decline including reduced DNA repair, mitochondrial dysfunction, and glucose intolerance (de Picciotto et al.”

“Increasing SIRT1 activity has been thought to improve endurance capacity by increasing mitochondrial function (Bai et al., 2011a, 2011b; Baur et al., 2006; Cantó et al., 2009; Lagouge et al., 2006; Pearson et al., 2008).”

“Tie2-Cre mouse was crossed to SIRT1f/f or SIRT1STOP mouse to generate ESKO (Tie2-Cre;SIRT1f/f) (Cheng et al., 2003; Potente et al., 2007) or ESTO (Tie2-Cre;SIRT1STOP) mouse, respectively.”

“SIRT1 is amember of the sirtuin family of NAD-dependent deacylases that mediate the health benefits of dietary restriction (DR) and can extend lifespan when overexpressed (Guarente, 2013; Haigis and Sinclair, 2010; Kanfi et al., 2012; Satoh et al., 2013).”

“MLECs were cultured in the presence of conditioned media (CM) with angiogenic factors provided by peroxisome proliferator- activated receptor g coactivator 1-a (PGC-1a)-overexpressing C2C12 myotubes (Arany et al., 2008).”

“Mice treated with NMN or NR for extended periods show no evidence of increased tumor burden (Mills et al., 2016; Zhang et al., 2016).”

“NAD Reverses Loss of the Microvasculature and Exercise Capacity in Old Mice via SIRT1 During aging, the level of NAD declines in many tissues, in part, due to increased activity of the NAD-consuming enzymes CD38 and PARP1 (Bai et al., 2011b; Camacho-Pereira et al., 2016; Li et al., 2017; Mouchiroud et al., 2013).”

“…is required for ischemia-induced neovascularization (Potente et al., 2007), vascular relaxation 74 Cell 173, 74–89, March 22, 2018 ª 2018 Elsevier Inc. (Mattagajasingh et al., 2007; Pearson et al., 2008; Ungvari et al., 2010), and is implicated in EC senescence (Ota et al., 2007; Zu et al., 2010).”

“Permeabilized fiber respiration Permeabilized fibers were prepared and mitochondrial function was analyzed according to published protocols with some modifications (Kuznetsov et al., 2008).”

“During aging, the level of NAD+ declines in many tissues, in part, due to increased activity of the NAD-consuming enzymes CD38 and PARP1 (Bai et al., 2011b; Camacho-Pereira et al., 2016; Li et al., 2017; Mouchiroud et al., 2013).”

“Exercise is currently the best way to delay the effects of aging on the microvasculature by promoting neovascularization, but little is known about why tissues become desensitized to exercise with age (Bassel-Duby and Olson, 2006; Booth and Thomason, 1991; Hood, 2001).”

“SIRT1 is amember of the sirtuin family of NAD-dependent deacylases that mediate the health benefits of dietary restriction (DR) and can extend lifespan when overexpressed (Guarente, 2013; Haigis and Sinclair, 2010; Kanfi et al., 2012; Satoh et al., 2013).”

“NAD precursors are an effective way to raise intracellular NAD+ levels and stimulate SIRT1 activity (Bogan and Brenner, 2008; Gomes et al., 2013; Yoshino et al., 2011).”

“Exercise is currently the best way to delay the effects of aging on the microvasculature by promoting neovascularization, but little is known about why tissues become desensitized to exercise with age (Bassel-Duby and Olson, 2006; Booth and Thomason, 1991; Hood, 2001).”

“In vertebrates, the Notch signaling pathway is indispensable for blood vessel formation (Blanco and Gerhardt, 2013).”

“In young muscle, SIRT1 is required for ischemia-induced neovascularization (Potente et al., 2007), vascular relaxation 74 Cell 173, 74–89, March 22, 2018 ª 2018 Elsevier Inc. (Mattagajasingh et al., 2007; Pearson et al., 2008; Ungvari et al., 2010), and is implicated in EC senescence (Ota et al.,…”

“REAGENT or RESOURCE SOURCE IDENTIFIER Methocel (viscosity: 4000 cps) Sigma-Aldrich Cat#M0512 BS1 lectin-FITC Sigma-Aldrich Cat#9381 Fetal Bovine Serum (FBS) Life Technologies Cat#10099141 Penicillin/Streptomycin (10,000 U/mL) Life Technologies Cat#15140122 Cytochrome c Oxidase from bovine heart Sigma-Aldrich Cat#C-549…”

“…is required for ischemia-induced neovascularization (Potente et al., 2007), vascular relaxation 74 Cell 173, 74–89, March 22, 2018 ª 2018 Elsevier Inc. (Mattagajasingh et al., 2007; Pearson et al., 2008; Ungvari et al., 2010), and is implicated in EC senescence (Ota et al., 2007; Zu et al., 2010).”

“SIRT1-activating compounds (STACs) such as resveratrol and SRT1720 have been pursued as a strategy for ameliorating agerelated diseases (Hubbard et al., 2013; Sinclair and Guarente, 2014).”

“Spheroid assay EC spheroids were generated as described previously (Korff and Augustin, 1998).”

“Aortic ring assay Aortic ring assay was performed according to the published protocol (Baker et al., 2011).”

“SIRT1 and H2S protect against oxidative stress-mediated apoptosis and senescence (Miller and Roth, 2007; Salminen et al., 2013; Suo et al., 2013; Wu et al., 2015).”

“SIRT1 is amember of the sirtuin family of NAD-dependent deacylases that mediate the health benefits of dietary restriction (DR) and can extend lifespan when overexpressed (Guarente, 2013; Haigis and Sinclair, 2010; Kanfi et al., 2012; Satoh et al., 2013).”

“…is required for ischemia-induced neovascularization (Potente et al., 2007), vascular relaxation 74 Cell 173, 74–89, March 22, 2018 ª 2018 Elsevier Inc. (Mattagajasingh et al., 2007; Pearson et al., 2008; Ungvari et al., 2010), and is implicated in EC senescence (Ota et al., 2007; Zu et al., 2010).”

“Hindlimb ischemia The effect of NMN in ischemia-induced vessel formation was assessed in a murine model of hindlimb ischemia using 8-month old SIRT1-iKO andWTmice according to the published protocol (Limbourg et al., 2009).”

“…2014), improve the exercise capacity of young mice (Cantó et al., 2012), and protect against age-related physiological decline including reduced DNA repair, mitochondrial dysfunction, and glucose intolerance (de Picciotto et al., 2016; Gomes et al., 2013; Li et al., 2017; Yoshino et al., 2011).”

“Dll4 coating was performed as described before (Guarani et al., 2011).”

“Indeed, mice lacking PGC-1a have limited angiogenesis after exercise (Chinsomboon et al., 2009) whereas mice overexpressing PGC-1a in muscle have increased mitochondria and capillary density (Lin et al.”

“It increases SIRT1 activity, protects against oxidative stress, and can promote angiogenesis (Longchamp et al., 2018).”

“A more recent approach has been to restore NAD levels by treating with NAD precursors such as nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN) (Cantó et al., 2015; Gomes et al., 2013; Mouchiroud et al., 2013; Ramsey et al., 2008).”

“…is required for ischemia-induced neovascularization (Potente et al., 2007), vascular relaxation 74 Cell 173, 74–89, March 22, 2018 ª 2018 Elsevier Inc. (Mattagajasingh et al., 2007; Pearson et al., 2008; Ungvari et al., 2010), and is implicated in EC senescence (Ota et al., 2007; Zu et al., 2010).”

“…The Jackson Laboratory JAX:007676 Mouse: C57BL/6J Myog-Cre Gift from Dr. Eric N. Olson, UT Southwestern TX Li et al., 2005 Mouse: C57BL/6J MCK-PGC-1a Gift from Dr. Bruce Spiegelman, Dana Farber Cancer Center MA Lin et al., 2002 Oligonucleotides Sir2A6860…”

“During aging, the level of NAD+ declines in many tissues, in part, due to increased activity of the NAD-consuming enzymes CD38 and PARP1 (Bai et al., 2011b; Camacho-Pereira et al., 2016; Li et al., 2017; Mouchiroud et al., 2013).”

“SIRT1-activating compounds (STACs) such as resveratrol and SRT1720 have been pursued as a strategy for ameliorating agerelated diseases (Hubbard et al., 2013; Sinclair and Guarente, 2014).”

“For reasons that are unclear, as we age there is an increase in muscle EC apoptosis, decreased neovascularization, and blood vessel loss (Groen et al., 2014; Wang et al., 2014), resulting in reduced muscle mass (sarcopenia) and a decline in strength and endurance in the later decades of life, even with exercise (Denis…”

“SIRT1 and H2S protect against oxidative stress-mediated apoptosis and senescence (Miller and Roth, 2007; Salminen et al., 2013; Suo et al., 2013; Wu et al., 2015).”

“NMN treatment induced proliferation (Figure S5L) and reduced apoptosis in ECs in a SIRT1-dependent manner, consistent with an inhibition of Notch (Noseda et al., 2004) (Figure S5M).”

“During aging, the level of NAD+ declines in many tissues, in part, due to increased activity of the NAD-consuming enzymes CD38 and PARP1 (Bai et al., 2011b; Camacho-Pereira et al., 2016; Li et al., 2017; Mouchiroud et al., 2013).”

“, 2012), and protect against age-related physiological decline including reduced DNA repair, mitochondrial dysfunction, and glucose intolerance (de Picciotto et al., 2016; Gomes et al., 2013; Li et al., 2017; Yoshino et al., 2011).”

“SIRT3 and SIRT6 also increase the angiogenic potential of ECs in culture (Cardus et al., 2013; Wei et al., 2017).”

“The performance of organs and tissues is critically dependent on a functional microcapillary network that maintains a supply of oxygen, exchanges heat and nutrients, and removes waste products (Olfert et al., 2009).”

“, 2014), resulting in reduced muscle mass (sarcopenia) and a decline in strength and endurance in the later decades of life, even with exercise (Denis et al., 1986; Prior et al., 2016; Ryan et al., 2006).”

“Supernatants were used to determine the activity for citrate synthase (CS) and succinate dehydrogenase (SDH) as described previously (Turner et al., 2009).”

“, 2014), resulting in reduced muscle mass (sarcopenia) and a decline in strength and endurance in the later decades of life, even with exercise (Denis et al., 1986; Prior et al., 2016; Ryan et al., 2006).”

“SIRT3 and SIRT6 also increase the angiogenic potential of ECs in culture (Cardus et al., 2013; Wei et al., 2017).”

“NAD precursors increase the angiogenic capacity of ECs in cell culture (Borradaile and Pickering, 2009; HughesLarge et al., 2014), improve the exercise capacity of young mice (Cantó et al.”

“In all cases, otherwise indicated, animals were administered NMN (400 mg/kg/day), NaHS (20 mg/kg/day) and/or GYY4137 (20mg/kg/day) (Rose et al., 2015) via drinking water at pH 7.2.”

“For reasons that are unclear, as we age there is an increase in muscle EC apoptosis, decreased neovascularization, and blood vessel loss (Groen et al., 2014; Wang et al., 2014), resulting in reduced muscle mass (sarcopenia) and a decline in strength and endurance in the later decades of life, even with exercise (Denis…”

“Alternatively, NAD levels in muscle and liver weremeasured by assay in-house developedmethod (Uddin et al., 2016).”

“In mammals, ectopic treatment with H2S induces SIRT1 in response to oxidative stress (Suo et al., 2013; Wu et al., 2015; Zheng et al., 2014) and protects rat hearts from ischemia/reperfusion via a mechanism requiring SIRT1 (Hu et al., 2016).”

“NMN did not alter the capillarity or exercise capacity of sedentary animals younger than 12 months (not shown), consistent with overexpression of Nampt, an NAD biosynthetic gene (Frederick et al., 2015).”

“Cell 173, 74–89, March 22, 2018 75 (legend on next page) 76 Cell 173, 74–89, March 22, 2018 Another DRmimetic is hydrogen sulfide (H2S), a gas generated endogenously by cystathionine b-synthase (CBS) and/or cystathionine g-lyase (CSE) (Hine and Mitchell, 2015).”

“In mammals, ectopic treatment with H2S induces SIRT1 in response to oxidative stress (Suo et al., 2013; Wu et al., 2015; Zheng et al., 2014) and protects rat hearts from ischemia/reperfusion via a mechanism requiring SIRT1 (Hu et al., 2016).”

“Cytochrome c oxidase staining of muscle sections was performed according to the published protocol (Ross, 2011).”

“The animals were then placed horizontally in the imaging chamber of MSOT inVision 256-TF small animal imaging system and three-dimensional scanning of the lower portion was performed as per manufacture’s protocol (Morscher et al., 2014).”

“From the age of 40 onward, tissue perfusion declines steadily and seemingly inexorably, leading to organ dysfunction and increasing frailty in the last decades of life (Degens, 1998; Le Couteur and Lakatta, 2010).”

“Despite the importance of capillary loss to human health, it is surprising how little we understand about its underlying causes (Le Couteur and Lakatta, 2010).”

“2-GW/EmGFP-miR negative control plasmid Life Technologies Cat#K4936-00 Lenti-VE-Cad miRNA plasmid Gift from Dr. Patty J. Lee (Yale University) Zhang et al., 2013 Software and Algorithms Adobe Creative Suite Adobe https://www.adobe.com FlowJo v10 FlowJo https://www.flowjo.com/ GraphPad Prism v7…”

“In mammals, ectopic treatment with H2S induces SIRT1 in response to oxidative stress (Suo et al., 2013; Wu et al., 2015; Zheng et al., 2014) and protects rat hearts from ischemia/reperfusion via a mechanism requiring SIRT1 (Hu et al., 2016).”

“Rotarod test Motor coordination was assessed using the rotarod test as previously described (Mersmann et al., 2011).”

“The difference in maximum and minimum video intensity was determined as the peak enhancement (Baltgalvis et al., 2014).”

“In mammals, ectopic treatment with H2S induces SIRT1 in response to oxidative stress (Suo et al., 2013; Wu et al., 2015; Zheng et al., 2014) and protects rat hearts from ischemia/reperfusion via a mechanism requiring SIRT1 (Hu et al., 2016).”

“A potential candidate is CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), an inhibitor of hypoxia-inducible factor 1 (HIF-1a) signaling that is implicated in type 2 diabetes (Wang et al., 2016).”

“Mounting evidence supports a critical role for NAD+ in stem cell rejuvenation, including in HSCs, endothelial stem cells, muscle stem cells, and melanocyte stem cells (Das et al., 2018; Vannini et al., 2019; Zhang et al., 2016).”