FTO Stabilizes MIS12 to Inhibit Vascular Smooth Muscle Cell Senescence in Atherosclerotic Plaque

AUTHORS

Sun J, Wang M, Jia F, Song J, Ren J, Hu B

ABSTRACT

Purpose: Atherosclerosis is the main cause of atherosclerotic cardiovascular disease (CVD). Here, we aimed to uncover the role and mechanisms of fat mass and obesity-associated genes (FTO) in the regulation of vascular smooth muscle cell (VSMC) senescence in atherosclerotic plaques.

Methods: ApoE−/− mice fed a high-fat diet (HFD) were used to establish an atherosclerotic animal model. Immunohistochemistry, and the staining of hematoxylin-eosin, Oil Red O, Sirius red, and Masson were performed to confirm the role of FTO in atherosclerosis in vivo. Subsequently, FTO expression in primary VSMCs is either upregulated or downregulated. Oxidized low-density lipoprotein (ox-LDL) was used to treat VSMCs, followed by EdU staining, flow cytometry, senescence-associated β-galactosidase (SA-β-gal) staining, immunofluorescence, telomere detection, RT-qPCR, and Western blotting to determine the molecular mechanisms by which FTO inhibits VSMC senescence.

Results: Decreased FTO expression was observed in progressive atherosclerotic plaques of ApoE−/− mice fed with HFD. FTO upregulation inhibits atherosclerotic lesions in mice. FTO inhibits VSMC aging in atherosclerotic plaques by helping VSMC withstand ox-LDL-induced cell cycle arrest and senescence. This process is achieved by stabilizing the MIS12 protein in VSMC through a proteasome-mediated pathway.

Conclusion: FTO inhibits VSMC senescence and subsequently slows the progression of atherosclerotic plaques by stabilizing the MIS12 protein.

Long noncoding RNA Malat1 protects against osteoporosis and bone metastasis

AUTHORS

Yang Zhao, Jingyuan Ning, Hongqi Teng, Yalan Deng, Marisela Sheldon, Lei Shi, Consuelo Martinez, Jie Zhang, Annie Tian, Yutong Sun, Shinichi Nakagawa, Fan Yao, Hai Wang & Li Ma

ABSTRACT

MALAT1, one of the few highly conserved nuclear long noncoding RNAs (lncRNAs), is abundantly expressed in normal tissues. Previously, targeted inactivation and genetic rescue experiments identified MALAT1 as a suppressor of breast cancer lung metastasis. On the other hand, Malat1-knockout mice are viable and develop normally. On a quest to discover the fundamental roles of MALAT1 in physiological and pathological processes, we find that this lncRNA is downregulated during osteoclastogenesis in humans and mice. Remarkably, Malat1 deficiency in mice promotes osteoporosis and bone metastasis of melanoma and mammary tumor cells, which can be rescued by genetic add-back of Malat1. Mechanistically, Malat1 binds to Tead3 protein, a macrophage-osteoclast–specific Tead family member, blocking Tead3 from binding and activating Nfatc1, a master regulator of osteoclastogenesis, which results in the inhibition of Nfatc1-mediated gene transcription and osteoclast differentiation. Notably, single-cell transcriptome analysis of clinical bone samples reveals that reduced MALAT1 expression in pre-osteoclasts and osteoclasts is associated with osteoporosis and metastatic bone lesions. Altogether, these findings identify Malat1 as a lncRNA that protects against osteoporosis and bone metastasis.

Piezo1 expression in chondrocytes controls endochondral ossification and osteoarthritis development

AUTHORS

Laura J. Brylka, Assil-Ramin Alimy, Miriam E. A. Tschaffon-Müller, Shan Jiang, Tobias Malte Ballhause, Anke Baranowsky, Simon von Kroge, Julian Delsmann, Eva Pawlus, Kian Eghbalian, Klaus Püschel, Astrid Schoppa, Melanie Haffner-Luntzer, David J. Beech, Frank Timo Beil, Michael Amling, Johannes Keller, Anita Ignatius, Timur A. Yorgan, Tim Rolvien & Thorsten Schinke

ABSTRACT

Piezo proteins are mechanically activated ion channels, which are required for mechanosensing functions in a variety of cell types. While we and others have previously demonstrated that the expression of Piezo1 in osteoblast lineage cells is essential for bone-anabolic processes, there was only suggestive evidence indicating a role of Piezo1 and/or Piezo2 in cartilage. Here we addressed the question if and how chondrocyte expression of the mechanosensitive proteins Piezo1 or Piezo2 controls physiological endochondral ossification and pathological osteoarthritis (OA) development. Mice with chondrocyte-specific inactivation of Piezo1 (Piezo1Col2a1Cre), but not of Piezo2, developed a near absence of trabecular bone below the chondrogenic growth plate postnatally. Moreover, all Piezo1Col2a1Cre animals displayed multiple fractures of rib bones at 7 days of age, which were located close to the growth plates. While skeletal growth was only mildly affected in these mice, OA pathologies were markedly less pronounced compared to littermate controls at 60 weeks of age. Likewise, when OA was induced by anterior cruciate ligament transection, only the chondrocyte inactivation of Piezo1, not of Piezo2, resulted in attenuated articular cartilage degeneration. Importantly, osteophyte formation and maturation were also reduced in Piezo1Col2a1Cre mice. We further observed increased Piezo1 protein abundance in cartilaginous zones of human osteophytes. Finally, we identified Ptgs2 and Ccn2 as potentially relevant Piezo1 downstream genes in chondrocytes. Collectively, our data do not only demonstrate that Piezo1 is a critical regulator of physiological and pathological endochondral ossification processes, but also suggest that Piezo1 antagonists may be established as a novel approach to limit osteophyte formation in OA.

A novel mouse model for familial hypocalciuric hypercalcemia (FHH1) reveals PTH-dependent and independent CaSR defects

AUTHORS

Catharina J. Küng, Arezoo Daryadel, Rocio Fuente, Betül Haykir, Martin Hrabĕ de Angelis, Nati Hernando, Isabel Rubio-Aliaga & Carsten A. Wagner

ABSTRACT

The Calcium-sensing receptor (CaSR) senses extracellular calcium, regulates parathyroid hormone (PTH) secretion, and has additional functions in various organs related to systemic and local calcium and mineral homeostasis. Familial hypocalciuric hypercalcemia type I (FHH1) is caused by heterozygous loss-of-function mutations in the CaSR gene, and is characterized by the combination of hypercalcemia, hypocalciuria, normal to elevated PTH, and facultatively hypermagnesemia and mild bone mineralization defects. To date, only heterozygous Casr null mice have been available as model for FHH1. Here we present a novel mouse FHH1 model identified in a large ENU-screen that carries an c.2579 T > A (p.Ile859Asn) variant in the Casr gene (CasrBCH002 mice). In order to dissect direct effects of the genetic variant from PTH-dependent effects, we crossed CasrBCH002 mice with PTH deficient mice. Heterozygous CasrBCH002 mice were fertile, had normal growth and body weight, were hypercalcemic and hypermagnesemic with inappropriately normal PTH levels and urinary calcium excretion replicating some features of FHH1. Hypercalcemia and hypermagnesemia were independent from PTH and correlated with higher expression of claudin 16 and 19 in kidneys. Likewise, reduced expression of the renal TRPM6 channel in CasrBCH002 mice was not dependent on PTH. In bone, mutations in Casr rescued the bone phenotype observed in Pth null mice by increasing osteoclast numbers and improving the columnar pattern of chondrocytes in the growth zone. In summary, CasrBCH002 mice represent a new model to study FHH1 and our results indicate that only a part of the phenotype is driven by PTH.

A novel micro-CT analysis for evaluating the regenerative potential of bone augmentation xenografts in rabbit calvarias

AUTHORS

Ilan Beitlitum, Fatma Rayyan, Ariel Pokhojaev, Haim Tal & Rachel Sarig

ABSTRACT

Guided Bone Regeneration is a common procedure, yet, as new grafting materials are being introduced into the market, a reliable evaluation method is required. Critical size defect in animal models provides an accurate simulation, followed by histological sections to evaluate the new bone formation. However, histology is destructive, two-dimensional and technique-sensitive. In this study we developed a novel volumetric Micro-CT analysis to quantify new bone formation characteristics. Eight adult female New Zealand white rabbits were subjected to calvarial critical-size defects. Four 8 mm in diameter circular defects were preformed in each animal, to allow random allocation of four treatment modalities. All calvarias were scanned using Micro-CT. Each defect was segmented into four equal parts: pristine bone, outer, middle, and inner. Amira software (v. 6.3, www.fei.com) was used to calculate the new bone volume in each region and compare it to that of the pristine bone. All grafting materials demonstrated that new bone formation decreased as it moved inward. Only the inner region differed across grafting materials (p = 0.001). The new Micro-CT analysis allowed us to divide each defect into 3D regions providing better understanding of the bone formation process. Amongst the various advantages of the Micro-CT, it enables us to quantify the graft materials and the newly formed bone independently, and to describe the defect morphology in 3D (bi- vs. uni-cortical defects). Providing an insight into the inner region of the defect can better predict the regenerative potential of the bone augmentation graft material. Therefore, the suggested Micro-CT analysis is beneficial for further developing of clinical approaches.

Musculoskeletal Health Worsened from Carnitine Supplementation and Not Impacted by a Novel Individualized Treadmill Training Protocol

AUTHORS

Ashley D. Troutman, Shruthi Srinivasan, Corinne E. Metzger, Paul B. Fallen, Neal Chen, Kalisha D. O'Neill, Matthew R. Allen, Annabel Biruete, Sharon M. Moe, Keith G. Avin

ABSTRACT

Introduction: Chronic kidney disease (CKD) negatively affects musculoskeletal health, leading to reduced mobility, and quality of life. In healthy populations, carnitine supplementation and aerobic exercise have been reported to improve musculoskeletal health. However, there are inconclusive results regarding their effectiveness and safety in CKD. We hypothesized that carnitine supplementation and individualized treadmill exercise would improve musculoskeletal health in CKD. Methods: We used a spontaneously progressive CKD rat model (Cy/+ rat) (n = 11–12/gr): (1) Cy/+ (CKD-Ctrl), (2) CKD-carnitine (CKD-Carn), and (3) CKD-treadmill (CKD-TM). Carnitine (250 mg/kg) was injected daily for 10 weeks. Rats in the treadmill group ran 4 days/week on a 5° incline for 10 weeks progressing from 30 min/day for week one to 40 min/day for week two to 50 min/day for the remaining 8 weeks. At 32 weeks of age, we assessed overall cardiopulmonary fitness, muscle function, bone histology and architecture, and kidney function. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons tests. Results: Moderate to severe CKD was confirmed by biochemistries for blood urea nitrogen (mean 43 ± 5 mg/dL CKD-Ctrl), phosphorus (mean 8 ± 1 mg/dL CKD-Ctrl), parathyroid hormone (PTH; mean 625 ± 185 pg/mL CKD-Ctrl), and serum creatinine (mean 1.1 ± 0.2 mg/mL CKD-Ctrl). Carnitine worsened phosphorous (mean 11 ± 3 mg/dL CKD-Carn; p < 0.0001), PTH (mean 1,738 ± 1,233 pg/mL CKD-Carn; p < 0.0001), creatinine (mean 1 ± 0.3 mg/dL CKD-Carn; p < 0.0001), cortical bone thickness (mean 0.5 ± 0.1 mm CKD-Ctrl, 0.4 ± 0.1 mm CKD-Carn; p < 0.05). Treadmill running significantly improves maximal aerobic capacity when compared to CKD-Ctrl (mean 14 ± 2 min CKD-TM, 10 ± 2 min CKD-Ctrl; p < 0.01). Conclusion: Carnitine supplementation worsened CKD progression, mineral metabolism biochemistries, and cortical porosity and did not have an impact on physical function. Individualized treadmill running improved maximal aerobic capacity but did not have an impact on CKD progression or bone properties. Future studies should seek to better understand carnitine doses in conditions of compromised renal function to prevent toxicity which may result from elevated carnitine levels and to optimize exercise prescriptions for musculoskeletal health.