Cardiac Left Ventricular miRNA-26a Is Downregulated in Ovariectomized Mice, Upregulated upon 17-Beta Estradiol Replacement, and Inversely Correlated with Collagen Type 1 Gene Expression.

Heart failure with preserved ejection fraction (HFpEF) is more prevalent in post- compared to pre-menopausal women. The underlying mechanisms are not fully understood. Data in humans is confounded by age and co-morbidities. We investigated the effects of ovariectomy and estrogen replacement on the left ventricular (LV) gene expression of pro-inflammatory and pro-fibrotic factors involved in HFpEF and putative regulating miRNAs. Nine-week-old C57BL/6 female mice were subjected to ovariectomy (OVX) or SHAM operation. OVX and SHAM groups were sacrificed 1-, 6-, and 12-weeks post-surgery (T1/SHAM; T1/OVX; T6/SHAM; T6/OVX, T12/SHAM). 17ß-estradiol (E2) or vehicle (VEH) was then administered to the OVX groups for 6 weeks (T12/OVX/E2; T12/OVX/VEH). Another SHAM group was sacrificed 12-weeks post-surgery. RNA and miRNAs were extracted from the LV apex. An early 3-fold increase in the gene expression of IL-1α, IL-6, Mmp9, Mmp12, Col1α1, and Col3α1 was observed one-week post-surgery in T1/OVX vs. T1/SHAM, but not at later time points. miRNA-26a was lower in T1/OVX vs. T1/SHAM and was inversely correlated with Col1α1 and Col3α1 expression 1-week post-surgery (r = -0.79 p < 0.001; r = -0.6 p = 0.007). miRNAs-26a, 29b, and 133a were significantly higher, while Col1α1, Col3α1, IL-1α, IL-6, Tnfα, Mmp12, and FasL gene expression was significantly lower in E2- compared to vehicle-treated OVX mice. miRNA-26a was inversely correlated with Col3α1 in T12/OVX/ E2 (r = -0.56 p = 0.02). OVX triggered an early increase in the gene expression of pro-inflammatory and pro-fibrotic factors, highlighting the importance of the early phase post-cessation of ovarian function. E2 replacement therapy, even if it was not immediately initiated after OVX, reversed these unfavorable changes and upregulated cardiac miRNA-26a, previously unknown to be affected by menopausal status.

RNA Sequencing Reveals Unique Transcriptomic Signatures of the Thyroid in a Murine Lung Cancer Model Treated with PD-1 and PD-L1 Antibodies.

Immune checkpoint inhibitors (ICI) are commonly associated with thyroid immune-related adverse events, yet the mechanism has not been fully elucidated. We aimed to further explore the mechanism of ICI-induced thyroid dysfunction by assessing changes induced in the thyroid transcriptome by ICI treatment (αPD-1/αPD-L1) in a lung cancer murine model. RNA-sequencing of thyroid tissues revealed 952 differentially expressed genes (DEGs) with αPD-1 treatment (|fold-change| ≥1.8, FDR < 0.05). Only 35 DEG were identified with αPD-L1, and we therefore focused on the αPD-1 group alone. Ingenuity Pathway Analysis revealed that of 952 DEGs with αPD-1 treatment, 362 were associated with functions of cell death and survival, with predicated activation of pathways for apoptosis and necrosis (Z = 2.89 and Z = 3.21, respectively) and negative activation of pathways for cell viability and cell survival (Z = -6.22 and Z = -6.45, respectively). Compared to previously published datasets of interleukin-1ß and interferon γ-treated human thyroid cells, apoptosis pathways were similarly activated. However, unique changes related to organ inflammation and upstream regulation by cytokines were observed. Our data suggest that there are unique changes in gene expression in the thyroid associated with αPD-1 therapy. ICI-induced thyroid dysfunction may be mediated by increased tissue apoptosis resulting in destructive thyroiditis.

Hepatic adropin is regulated by estrogen and contributes to adverse metabolic phenotypes in ovariectomized mice.

OBJECTIVE: Menopause is associated with visceral adiposity, hepatic steatosis and increased risk for cardiovascular disease. As estrogen replacement therapy is not suitable for all postmenopausal women, a need for alternative therapeutics and biomarkers has emerged. METHODS: 9-week-old C57BL/6 J female mice were subjected to ovariectomy (OVX) or SHAM surgery (n = 10 per group), fed a standard diet and sacrificed 6- & 12 weeks post-surgery. RESULTS: Increased weight gain, hepatic triglyceride content and changes in hepatic gene expression of Cyp17a1, Rgs16, Fitm1 as well as Il18, Rares2, Retn, Rbp4 in mesenteric visceral adipose tissue (VAT) were observed in OVX vs. SHAM. Liver RNA-sequencing 6-weeks post-surgery revealed changes in genes and microRNAs involved in fat metabolism in OVX vs. SHAM mice. Energy Homeostasis Associated gene (Enho) coding for the hepatokine adropin was significantly reduced in OVX mice livers and strongly inversely correlated with weight gain (r = -0.7 p < 0.001) and liver triglyceride content (r = -0.4, p = 0.04), with a similar trend for serum adropin. In vitro, Enho expression was tripled by 17ß-estradiol in BNL 1 ME liver cells with increased adropin in supernatant. Analysis of open-access datasets revealed increased hepatic Enho expression in estrogen treated OVX mice and estrogen dependent ERα binding to Enho. Treatment of 5-month-old OVX mice with Adropin (i.p. 450 nmol/kg/twice daily, n = 4,5 per group) for 6-weeks reversed adverse adipokine gene expression signature in VAT, with a trended increase in lean body mass and decreased liver TG content with upregulation of Rgs16. CONCLUSIONS: OVX is sufficient to induce deranged metabolism in adult female mice. Hepatic adropin is regulated by estrogen, negatively correlated with adverse OVX-induced metabolic phenotypes, which were partially reversed with adropin treatment. Adropin should be further explored as a potential therapeutic target and biomarker for menopause-related metabolic derangement.

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 (Sirt1 Δ/+) and wild type (WT) mice. Decreased expression of the thermogenic genes: Prdm16, Pgc1α, Foxc2, Dio2, and ß3AR was detected in whole tibiae derived from Sirt1 Δ/+ 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 Sirt1 Δ/+ 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 ß3AR, 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.

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.

The Sirtuin1 activator SRT3025 down-regulates sclerostin and rescues ovariectomy-induced bone loss and biomechanical deterioration in female mice.

Estrogen deficiency leads to rapid bone loss and skeletal fragility. Sclerostin, encoded by the sost gene, and a product of the osteocyte, is a negative regulator of bone formation. Blocking sclerostin increases bone mass and strength in animals and humans. Sirtuin1 (Sirt1), a player in aging and metabolism, regulates bone mass and inhibits sost expression by deacetylating histone 3 at its promoter. We asked whether a Sirt1-activating compound could rescue ovariectomy (OVX)-induced bone loss and biomechanical deterioration in 9-week-old C57BL/6 mice. OVX resulted in a substantial decrease in skeletal Sirt1 expression accompanied by an increase in sclerostin. Oral administration of SRT3025, a Sirt1 activator, at 50 and 100 mg/kg·d for 6 weeks starting 6 weeks after OVX fully reversed the deleterious effects of OVX on vertebral bone mass, microarchitecture, and femoral biomechanical properties. Treatment with SRT3025 decreased bone sclerostin expression and increased cortical periosteal mineralizing surface and serum propeptide of type I procollagen, a bone formation marker. In vitro, in the murine long bone osteocyte-Y4 osteocyte-like cell line SRT3025 down-regulated sclerostin and inactive ß-catenin, whereas a reciprocal effect was observed with EX-527, a Sirt1 inhibitor. Sirt1 activation by Sirt1-activating compounds is a potential novel pathway to down-regulate sclerostin and design anabolic therapies for osteoporosis concurrently ameliorating other metabolic and age-associated conditions.

Sirt1 is a regulator of bone mass and a repressor of Sost encoding for sclerostin, a bone formation inhibitor.

Sirt1, the mammalian ortholog of the yeast Sir2 (silent information regulator 2), was shown to play an important role in metabolism and in age-associated diseases, but its role in skeletal homeostasis and osteoporosis has yet not been studied. Using 129/Sv mice with a germline mutation in the Sirt1 gene, we demonstrate that Sirt1 haplo-insufficient (Sirt1(+/-)) female mice exhibit a significant reduction in bone mass characterized by decreased bone formation and increased marrow adipogenesis. Importantly, we identify Sost, encoding for sclerostin, a critical inhibitor of bone formation, as a novel target of Sirt1. Using chromatin immunoprecipitation analysis, we reveal that Sirt1 directly and negatively regulates Sost gene expression by deacetylating histone 3 at lysine 9 at the Sost promoter. Sost down-regulation by small interfering RNA and the administration of a sclerostin-neutralizing antibody restore gene expression of osteocalcin and bone sialoprotein as well as mineralized nodule formation in Sirt1(+/-) marrow-derived mesenchymal stem cells induced to osteogenesis. These findings reveal a novel role for Sirt1 in bone as a regulator of bone mass and a repressor of sclerostin, and have potential implications suggesting that Sirt1 is a target for promoting bone formation as an anabolic approach for treatment of osteoporosis.

Pathogenicity and immunogenicity in mice of vaccinia viruses mutated in the viral envelope proteins A33R and B5R.

The pathogenicity and immunogenicity in mice of WR.cl and WR.c3, two mutants of the Western Reserve (WR) strain of vaccinia virus, mutated in the A33R and B5R proteins of the outer envelope of the virus, respectively, were studied. WR.c1 was the most attenuated virus, WR.c3 was somewhat more pathogenic, while WR was the most virulent of the three. While the WR and the WR.c3 viruses, intranasally inoculated into mice, spread efficiently to the different internal organs of the animal, including the brain, WR.c1 was restricted to the lungs only. Mice, intranasally infected with 500 plaque forming units of the WR, WR.c1, or WR.c3 viruses, were protected against infection with a lethal dose of the WR strain.