Browsing by Author "Aguilar-Perez, Alexandra"

Now showing 1 - 5 of 5
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    Age- and sex-dependent role of osteocytic pannexin1 on bone and muscle mass and strength
    (Nature Research, 2019-09-25) Aguilar-Perez, Alexandra; Pacheco-Costa, Rafael; Atkinson, Emily G.; Deosthale, Padmini; Davis, Hannah M.; Essex, Alyson L.; Dilley, Julian E.; Gomez, Leland; Rupert, Joseph E.; Zimmers, Teresa A.; Thompson, Roger J.; Allen, Matthew R.; Plotkin, Lilian I.; Anatomy and Cell Biology, School of Medicine
    Pannexins (Panxs), glycoproteins that oligomerize to form hemichannels on the cell membrane, are topologically similar to connexins, but do not form cell-to-cell gap junction channels. There are 3 members of the family, 1-3, with Panx1 being the most abundant. All Panxs are expressed in bone, but their role in bone cell biology is not completely understood. We now report that osteocytic Panx1 deletion (Panx1Δot) alters bone mass and strength in female mice. Bone mineral density after reaching skeletal maturity is higher in female Panx1Δot mice than in control Panx1fl/fl mice. Further, osteocytic Panx1 deletion partially prevented aging effects on cortical bone structure and mechanical properties. Young 4-month-old female Panx1Δot mice exhibited increased lean body mass, even though pannexin levels in skeletal muscle were not affected; whereas no difference in lean body mass was detected in male mice. Furthermore, female Panx1-deficient mice exhibited increased muscle mass without changes in strength, whereas Panx1Δot males showed unchanged muscle mass and decreased in vivo maximum plantarflexion torque, indicating reduced muscle strength. Our results suggest that osteocytic Panx1 deletion increases bone mass in young and old female mice and muscle mass in young female mice, but has deleterious effects on muscle strength only in males.
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    Kynurenine Inhibits Autophagy and Promotes Senescence in Aged Bone Marrow Mesenchymal Stem Cells Through the Aryl Hydrocarbon Receptor Pathway
    (Elsevier, 2020-02) Kondrikov, Dmitry; Elmansi, Ahmed; Bragg, Robert Tailor; Mobley, Tanner; Barrett, Thomas; Eisa, Nada; Kondrikova, Galina; Schoeinlein, Patricia; Aguilar-Perez, Alexandra; Shi, Xing-Ming; Fulzele, Sadanand; McGee Lawrence, Meghan; Hamrick, Mark; Isales, Carlos; Hill, William; Anatomy and Cell Biology, School of Medicine
    Osteoporosis is an age-related deterioration in bone health that is, at least in part, a stem cell disease. The different mechanisms and signaling pathways that change with age and contribute to the development of osteoporosis are being identified. One key upstream mechanism that appears to target a number of osteogenic pathways with age is kynurenine, a tryptophan metabolite and an endogenous Aryl hydrocarbon receptor (AhR) agonist. The AhR signaling pathway has been reported to promote aging phenotypes across species and in different tissues. We previously found that kynurenine accumulates with age in the plasma and various tissues including bone and induces bone loss and osteoporosis in mice. Bone marrow mesenchymal stem cells (BMSCs) are responsible for osteogenesis, adipogenesis, and overall bone regeneration. In the present study, we investigated the effect of kynurenine on BMSCs, with a focus on autophagy and senescence as two cellular processes that control BMSCs proliferation and differentiation capacity. We found that physiological levels of kynurenine (10 and 100 μM) disrupted autophagic flux as evidenced by the reduction of LC3B-II, and autophagolysosomal production, as well as a significant increase of p62 protein level. Additionally, Kynurenine also induced a senescent phenotype in BMSCs as shown by the increased expression of several senescence markers including senescence associated β-galactosidase in BMSCs. Additionally, western blotting reveals that levels of p21, another marker of senescence, also increased in kynurenine-treated BMSCs, while senescent-associated aggregation of nuclear H3K9me3 also showed a significant increase in response to kynurenine treatment. To validate that these effects are in fact due to AhR signaling pathway, we utilized two known AhR antagonists: CH-223191, and 3’,4’-Dimethoxyflavone to try to block AhR signaling and rescue kynurenine/AhR mediated effects. Indeed, AhR inhibition restored kynurenine-suppressed autophagy levels as shown by levels of LC3B-II, p62 and autophagolysosomal formation demonstrating a rescuing of autophagic flux. Furthermore, inhibition of AhR signaling prevented the kynurenine-induced increase in senescence associated β-galactosidase and p21 levels, as well as blocking aggregation of nuclear H3K9me3. Taken together, our results suggest that kynurenine inhibits autophagy and induces senescence in BMSCs via AhR signaling, and that this may be a novel target to prevent or reduce age-associated bone loss and osteoporosis.
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    Megakaryocytes promote osteoclastogenesis in aging
    (Impact Journals, 2020-07-07) Kanagasabapathy, Deepa; Blosser, Rachel J.; Maupin, Kevin A.; Hong, Jung Min; Alvarez, Marta; Ghosh, Joydeep; Mohamad, Safa F.; Aguilar-Perez, Alexandra; Srour, Edward F.; Kacena, Melissa A.; Bruzzaniti, Angela; Orthopaedic Surgery, School of Medicine
    Megakaryocytes (MKs) support bone formation by stimulating osteoblasts (OBs) and inhibiting osteoclasts (OCs). Aging results in higher bone resorption, leading to bone loss. Whereas previous studies showed the effects of aging on MK-mediated bone formation, the effects of aging on MK-mediated OC formation is poorly understood. Here we examined the effect of thrombopoietin (TPO) and MK-derived conditioned media (CM) from young (3-4 months) and aged (22-25 months) mice on OC precursors. Our findings showed that aging significantly increased OC formation in vitro. Moreover, the expression of the TPO receptor, Mpl, and circulating TPO levels were elevated in the bone marrow cavity. We previously showed that MKs from young mice secrete factors that inhibit OC differentiation. However, rather than inhibiting OC development, we found that MKs from aged mice promote OC formation. Interestingly, these age-related changes in MK functionality were only observed using female MKs, potentially implicating the sex steroid, estrogen, in signaling. Further, RANKL expression was highly elevated in aged MKs suggesting MK-derived RANKL signaling may promote osteoclastogenesis in aging. Taken together, these data suggest that modulation in TPO-Mpl expression in bone marrow and age-related changes in the MK secretome promote osteoclastogenesis to impact skeletal aging.
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    Neonatal Osteomacs and Bone Marrow Macrophages Differ in Phenotypic Marker Expression and Function
    (Wiley, 2021) Mohamad, Safa F.; Gunawan, Andrea; Blosser, Rachel; Childress, Paul; Aguilar-Perez, Alexandra; Ghosh, Joydeep; Hong, Jung Min; Liu, Jianyun; Kanagasabapathy, Deepa; Kacena, Melissa A.; Srour, Edward F.; Bruzzaniti, Angela; Medicine, School of Medicine
    Osteomacs (OM) are specialized bone-resident macrophages that are a component of the hematopoietic niche and support bone formation. Also located in the niche are a second subset of macrophages, namely bone marrow-derived macrophages (BM Mφ). We previously reported that a subpopulation of OM co-express both CD166 and CSF1R, the receptor for macrophage colony-stimulating factor (MCSF), and that OM form more bone-resorbing osteoclasts than BM Mφ. Reported here are single-cell quantitative RT-PCR (qRT-PCR), mass cytometry (CyTOF), and marker-specific functional studies that further identify differences between OM and BM Mφ from neonatal C57Bl/6 mice. Although OM express higher levels of CSF1R and MCSF, they do not respond to MCSF-induced proliferation, in contrast to BM Mφ. Moreover, receptor activator of NF-κB ligand (RANKL), without the addition of MCSF, was sufficient to induce osteoclast formation in OM but not BM Mφ cultures. OM express higher levels of CD166 than BM Mφ, and we found that osteoclast formation by CD166-/- OM was reduced compared with wild-type (WT) OM, whereas CD166-/- BM Mφ showed enhanced osteoclast formation. CD110/c-Mpl, the receptor for thrombopoietin (TPO), was also higher in OM, but TPO did not alter OM-derived osteoclast formation, whereas TPO stimulated BM Mφ osteoclast formation. CyTOF analyses demonstrated OM uniquely co-express CD86 and CD206, markers of M1 and M2 polarized macrophages, respectively. OM performed equivalent phagocytosis in response to LPS or IL-4/IL-10, which induce polarization to M1 and M2 subtypes, respectively, whereas BM Mφ were less competent at phagocytosis when polarized to the M2 subtype. Moreover, in contrast to BM Mφ, LPS treatment of OM led to the upregulation of CD80, an M1 marker, as well as IL-10 and IL-6, known anti-inflammatory cytokines. Overall, these data reveal that OM and BM Mφ are distinct subgroups of macrophages, whose phenotypic and functional differences in proliferation, phagocytosis, and osteoclast formation may contribute physiological specificity during health and disease.
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    Osteomacs interact with megakaryocytes and osteoblasts to regulate murine hematopoietic stem cell function
    (ASH, 2017-12) Mohamad, Safa F.; Xu, Linlin; Ghosh, Joydeep; Childress, Paul J.; Abeysekera, Irushi; Himes, Evan R.; Wu, Hao; Alvarez, Marta B.; Davis, Korbin M.; Aguilar-Perez, Alexandra; Hong, Jung Min; Bruzzaniti, Angela; Kacena, Melissa A.; Srour, Edward F.; Biomedical Sciences and Comprehensive Care, School of Dentistry
    Networking between hematopoietic stem cells (HSCs) and cells of the hematopoietic niche is critical for stem cell function and maintenance of the stem cell pool. We characterized calvariae-resident osteomacs (OMs) and their interaction with megakaryocytes to sustain HSC function and identified distinguishing properties between OMs and bone marrow (BM)–derived macrophages. OMs, identified as CD45+F4/80+ cells, were easily detectable (3%-5%) in neonatal calvarial cells. Coculture of neonatal calvarial cells with megakaryocytes for 7 days increased OM three- to sixfold, demonstrating that megakaryocytes regulate OM proliferation. OMs were required for the hematopoiesis-enhancing activity of osteoblasts, and this activity was augmented by megakaryocytes. Serial transplantation demonstrated that HSC repopulating potential was best maintained by in vitro cultures containing osteoblasts, OMs, and megakaryocytes. With or without megakaryocytes, BM-derived macrophages were unable to functionally substitute for neonatal calvarial cell–associated OMs. In addition, OMs differentiated into multinucleated, tartrate resistant acid phosphatase–positive osteoclasts capable of bone resorption. Nine-color flow cytometric analysis revealed that although BM-derived macrophages and OMs share many cell surface phenotypic similarities (CD45, F4/80, CD68, CD11b, Mac2, and Gr-1), only a subgroup of OMs coexpressed M-CSFR and CD166, thus providing a unique profile for OMs. CD169 was expressed by both OMs and BM-derived macrophages and therefore was not a distinguishing marker between these 2 cell types. These results demonstrate that OMs support HSC function and illustrate that megakaryocytes significantly augment the synergistic activity of osteoblasts and OMs. Furthermore, this report establishes for the first time that the crosstalk between OMs, osteoblasts, and megakaryocytes is a novel network supporting HSC function.