The Sirt1 Activators SRT2183 and SRT3025 Inhibit RANKL-Induced Osteoclastogenesis in Bone Marrow-Derived Macrophages and Down-Regulate Sirt3 in Sirt1 Null Cells.

Increased osteoclast-mediated bone resorption is characteristic of osteoporosis, malignant bone disease and inflammatory arthritis. Targeted deletion of Sirtuin1 (Sirt1), a key player in aging and metabolism, in osteoclasts results in increased osteoclast-mediated bone resorption in vivo, making it a potential novel therapeutic target to block bone resorption. Sirt1 activating compounds (STACs) were generated and were investigated in animal disease models and in humans however their mechanism of action was a source of controversy. We studied the effect of SRT2183 and SRT3025 on osteoclastogenesis in bone-marrow derived macrophages (BMMs) in vitro, and discovered that these STACs inhibit RANKL-induced osteoclast differentiation, fusion and resorptive capacity without affecting osteoclast survival. SRT2183 and SRT3025 activated AMPK, increased Sirt1 expression and decreased RelA/p65 lysine310 acetylation, critical for NF-κB activation, and an established Sirt1 target. However, inhibition of osteoclastogenesis by these STACs was also observed in BMMs derived from sirt1 knock out (sirt1-/-) mice lacking the Sirt1 protein, in which neither AMPK nor RelA/p65 lysine 310 acetylation was affected, confirming that these effects require Sirt1, but suggesting that Sirt1 is not essential for inhibition of osteoclastogenesis by these STACs under these conditions. In sirt1 null osteoclasts treated with SRT2183 or SRT3025 Sirt3 was found to be down-regulated. Our findings suggest that SRT2183 and SRT3025 activate Sirt1 and inhibit RANKL-induced osteoclastogenesis in vitro however under conditions of Sirt1 deficiency can affect Sirt3. As aging is associated with reduced Sirt1 level and activity, the influence of STACs on Sirt3 needs to be investigated in vivo in animal and human disease models of aging and osteoporosis

Sirt1 Promotes a Thermogenic Gene Program in Bone Marrow Adipocytes: From Mice to (Wo)Men

Bone marrow adipose tissue (MAT) is influenced by nutritional cues, and participates in whole body energy metabolism. To investigate the role of Sirtuin1 (Sirt1), a key player in metabolism, in MAT, marrow adiposity was evaluated in inbred 5-month-old 129/Sv Sirt1 haplo-insufficient (Sirt1Δ/+) and wild type (WT) mice. Decreased expression of the thermogenic genes: Prdm16, Pgc1α, Foxc2, Dio2, and β3AR was detected in whole tibiae derived from Sirt1Δ/+ compared to WT female mice. Similarly, decreased expression of Prdm16 and Pgc1α was observed in primary bone marrow mesenchymal stem cell (BM-MSC) cultures obtained from Sirt1Δ/+ compared to WT female mice, suggesting a cell autonomous effect of Sirt1 in BM-MSCs. In vitro, Sirt1 over-expression in the mesenchymal embryonic fibroblast stem cell line C3HT101/2 increased Pgc1α and Prdm16 protein level. Similarly, pharmacologic activation of Sirt1 by SRT3025 increased Foxc2, Pgc1α, Dio2, Tfam, and Cyc1 expression while inhibition of Sirt1 by EX527 down-regulated UCP1 in C3HT101/2 cells. Importantly, in human femoral BM-MSCs obtained from female patients undergoing hip operations for fracture or osteoarthritis, Sirt1 activation by SRT3025 increased PGC1α mRNA and protein level. Blocking sclerostin, an inhibitor of the WNT pathway and a Sirt1 target, by the monoclonal humanized antibody (Sc-AbII), stimulated β3AR, PRDM16, and UCP1 gene expression, and increased PGC1α protein level. These results show that Sirt1 stimulates a thermogenic gene program in marrow adipocytes in mice and humans via PGC1α activation and sclerostin inhibition. The implications of these findings to bone health, hematopoiesis and whole body energy metabolism remain to be investigated.ISSN:1664-239

SRT2183 inhibits RANKL-induced osteoclastogenesis and pit formation in s<i>irt1</i>^<i>-/-</i> BMMs.

<p>(A) Sirt1 expression in WT- and in <i>Sirt1</i>^<i>-/-</i>-derived osteoclasts. PCR amplification of exons 1–9 of the s<i>irt1</i> gene (left panel) and Western blot analysis with Sirt1 antibody (right panel) demonstrates complete loss of Sirt1 protein in osteoclasts obtained from <i>Sirt1</i>^<i>Δ/Δ</i> (<i>Sirt1</i>^<i>-/-</i>) mice. (B) The effect of SRT2183 on osteoclast differentiation in <i>Sirt1</i>^<i>-/-</i>-derived BMMs. BMMs were inducted to osteoclastogenesis with RANKL in the presence or absence of SRT2183. TRAP staining performed 4 days post induction. (C) The effect of SRT2183 on pit formation in <i>Sirt1</i>^<i>-/-</i>-derived BMMs stimulated with RANKL. An eroded area (left panel) and pit formation assay (right) are shown. (D) The effect of SRT2183 on p65 acetylation (Lys310). Western blot analysis of p65K310 ac and p65 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (E) The effect of SRT2183 on AMPKα phosphorylation (Thr172). Western blot analysis of pAMPKα and AMPKα in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (F) The effect of SRT2183 on IκBα protein level. Western blot analysis of IκBα and GAPDH in SRT2183- and vehicle-treated BMMs 24 hours post RANKL stimulation. (G-H) The effect of SRT2183 on Sirt3 protein (G) and gene expression (H). Western blot analysis of Sirt3 and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation (G). Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to <i>Polr2a</i> (H). (I) The effect of SRT2183 on superoxide dismutase 2 (Sod2) Lys68 acetylation. Western blot analysis of acetylated (ac) Sod2K68 and Sod2 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ± SEM (n = 3 independent experiments), analyzed by paired Student's <i>t</i>-test paired (C) or one-sample Student's <i>t</i>-test (H-I); ***<i>P</i><0.001, ****<i>P</i><0.0001, versus vehicle-treated BMMs. Magnification X40; scale bar 1mm.</p

SRT2183 inhibits RANKL-induced NFATc1 activation in bone marrow-derived macrophages (BMMs).

<p>(A) The effect of SRT2183 on NFATc1 protein level. Western blot analysis of NFATc1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. NFATc1- nuclear factor of activated T-cell cytoplasmic 1. (B) The effect of SRT2183 on DC-STAMP protein level. Western blot analysis of DC-STAMP and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. DC-STAMP- dendritic cell-specific transmembrane protein. (C) The effect of SRT2183 on mRNA expression of osteoclast markers and fusion-related genes. SRT2183 or vehicle were co-administrated with RANKL. Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to <i>GAPDH</i>. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's <i>t</i>-test, *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001 compared to vehicle-treated BMMs.</p

SRT2183 inhibits RANKL-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs).

<p>(A) Chemical structure of SRT2183. (B-C) TRAP staining of SRT2183 or vehicle-treated BMMs inducted to osteoclastogenesis 4 days post RANKL stimulation (B). Total number of osteoclasts (left panel) and number of multinuclear cells (right) are shown (C). (D) The effect of SRT2183 on pit formation by RANKL-induced BMMs. A pit formation assay (left panel) and eroded area (right) are shown. (E) Time course of the effect of SRT2183 or vehicle (DMSO) administration on osteoclast differentiation. SRT2183 or a vehicle was added in the proliferation (a), differentiation (b, c) or maturation (c, d) phase. Arrows indicate periods of treatments with SRT2183. TRAP staining is shown. (F) The effect of SRT2183 on cell proliferation during the proliferation phase. SRT2183 or a vehicle were co-administrated with M-CSF for 72 hours on day of plating. (G-H) The effect of SRT2183 on cell viability and apoptosis during the proliferation phase (G) and the differentiation and maturation phase (H). SRT2183 or vehicle were co-administrated with M-CSF for 72 hours on day of plating (G) or with RANKL 3 days post plating (H). The graphs illustrate fold change in Caspase 3 activity and the percent change in living cells with time. Data are Mean ±SEM (n = 3 independent experiments), analyzed by 2 way ANOVA with nuclei number and treatment as the independent variables followed by Sidak's post-hoc correction (C), paired Student's <i>t</i>-test (D), one-sample Student's <i>t</i>-test (F-H), *<i>P</i><0.05; **<i>P</i><0.01 compared to vehicle-treated BMMs. Magnification X40; Scale bar 1mm.</p

SRT2183 activates AMPK and deacetylates RelA/p65 K310 in RANKL-induced BMMs.

<p>(A) The effect of SRT2183 on AMPKα phosphorylation (Thr172). Western blot analysis of pAMPKα and AMPKα in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; AMPKα- AMP-activated protein kinase alpha. (B) The effect of SRT2183 on ACC phosphorylation. Western blot analysis of pACC and ACC in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; ACC-Acetyl CoA Carboxylase. (C) The effect of SRT2183 on Sirt1 protein level in RANKL-stimulated osteoclasts. Western blot analysis of Sirt1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (D) The effect of SRT2183 on IκBα protein level. Western blot analysis of IκBα and GAPDH in SRT2183- and vehicle-treated BMMs 24 hours post RANKL stimulation. (E) The effect of SRT2183 on p65 acetylation (Lys310). Western blot analysis of p65K310 ac and p65 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's <i>t</i>-test; *<i>P</i><0.05 versus vehicle-treated BMMs.</p

Ventilation and outcomes following robotic-assisted abdominal surgery : an international, multicentre observational study

Background: International data on the epidemiology, ventilation practice, and outcomes in patients undergoing abdominal robotic-assisted surgery (RAS) are lacking. The aim of the study was to assess the incidence of postoperative pulmonary complications (PPCs), and to describe ventilator management after abdominal RAS. Methods: This was an international, multicentre, prospective study in 34 centres in nine countries. Patients >= 18 yr of age undergoing abdominal RAS were enrolled between April 2017 and March 2019. The Assess Respiratory Risk in Surgical Patients in Catalonia (ARISCAT) score was used to stratify for higher risk of PPCs (>= 26). The primary outcome was the incidence of PPCs. Secondary endpoints included the preoperative risk for PPCs and ventilator management. Results: Of 1167 subjects screened, 905 abdominal RAS patients were included. Overall, 590 (65.2%) patients were at increased risk for PPCs. Meanwhile, 172 (19%) patients sustained PPCs, which occurred more frequently in 132 (22.4%) patients at increased risk, compared with 40 (12.7%) patients at lower risk of PPCs (absolute risk difference: 12.2% [95% confidence intervals (CI), 6.8-17.6%]; P<0.001). Plateau and driving pressures were higher in patients at increased risk, compared with patients at low risk of PPCs, but no ventilatory variables were independently associated with increased occurrence of PPCs. Development of PPCs was associated with a longer hospital stay. Conclusions: One in five patients developed one or more PPCs (chiefly unplanned oxygen requirement), which was associated with a longer hospital stay. No ventilatory variables were independently associated with PPCs

Effect of Intraoperative High Positive End-Expiratory Pressure (PEEP) With Recruitment Maneuvers vs Low PEEP on Postoperative Pulmonary Complications in Obese Patients: A Randomized Clinical Trial (vol 321, pg 2292, 2019)

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Effect of intraoperative high Positive End-Expiratory Pressure (PEEP) with recruitment maneuvers vs low PEEP on postoperative pulmonary complications in obese patients : a randomized clinical trial

IMPORTANCE An intraoperative higher level of positive end-expiratory positive pressure (PEEP) with alveolar recruitment maneuvers improves respiratory function in obese patients undergoing surgery, but the effect on clinical outcomes is uncertain. OBJECTIVE To determine whether a higher level of PEEP with alveolar recruitment maneuvers decreases postoperative pulmonary complications in obese patients undergoing surgery compared with a lower level of PEEP. DESIGN, SETTING, AND PARTICIPANTS Randomized clinical trial of 2013 adults with body mass indices of 35 or greater and substantial risk for postoperative pulmonary complications who were undergoing noncardiac, nonneurological surgery under general anesthesia. The trial was conducted at 77 sites in 23 countries from July 2014-February 2018; final follow-up: May 2018. INTERVENTIONS Patients were randomized to the high level of PEEP group (n = 989), consisting of a PEEP level of 12 cm H2O with alveolar recruitment maneuvers (a stepwise increase of tidal volume and eventually PEEP) or to the low level of PEEP group (n = 987), consisting of a PEEP level of 4 cm H2O. All patients received volume-controlled ventilation with a tidal volume of 7 mL/kg of predicted body weight. MAIN OUTCOMES AND MEASURES The primary outcomewas a composite of pulmonary complications within the first 5 postoperative days, including respiratory failure, acute respiratory distress syndrome, bronchospasm, new pulmonary infiltrates, pulmonary infection, aspiration pneumonitis, pleural effusion, atelectasis, cardiopulmonary edema, and pneumothorax. Among the 9 prespecified secondary outcomes, 3 were intraoperative complications, including hypoxemia (oxygen desaturation with SpO(2) 1 minute). RESULTS Among 2013 adults who were randomized, 1976 (98.2%) completed the trial (mean age, 48.8 years; 1381 [69.9%] women; 1778 [90.1%] underwent abdominal operations). In the intention-to-treat analysis, the primary outcome occurred in 211 of 989 patients (21.3%) in the high level of PEEP group compared with 233 of 987 patients (23.6%) in the low level of PEEP group (difference, -2.3%[95% CI, -5.9% to 1.4%]; risk ratio, 0.93 [95% CI, 0.83 to 1.04]; P =.23). Among the 9 prespecified secondary outcomes, 6 were not significantly different between the high and low level of PEEP groups, and 3 were significantly different, including fewer patients with hypoxemia (5.0% in the high level of PEEP group vs 13.6% in the low level of PEEP group; difference, -8.6%[95% CI, -11.1% to 6.1%]; P <.001). CONCLUSIONS AND RELEVANCE Among obese patients undergoing surgery under general anesthesia, an intraoperative mechanical ventilation strategy with a higher level of PEEP and alveolar recruitment maneuvers, compared with a strategy with a lower level of PEEP, did not reduce postoperative pulmonary complications

Effect of Intraoperative High Positive End-Expiratory Pressure (PEEP) With Recruitment Maneuvers vs Low PEEP on Postoperative Pulmonary Complications in Obese Patients: A Randomized Clinical Trial.

IMPORTANCE An intraoperative higher level of positive end-expiratory positive pressure (PEEP) with alveolar recruitment maneuvers improves respiratory function in obese patients undergoing surgery, but the effect on clinical outcomes is uncertain