The energetic costs of bone formation require osteoblasts to coordinate their activities with tissues, like adipose, that can supply energy-dense macronutrients. In the case of intermittent parathyroid hormone treatment (PTH), a strategy used to reduce fracture risk, bone formation is proceeded by a change in systemic lipid homeostasis. To investigate the requirement for fatty acid oxidation by osteoblasts during PTH-induced bone formation, we subjected mice with osteoblast-specific deficiency of mitochondrial long-chain β-oxidation as well as mice with adipocyte-specific deficiency for the PTH receptor or adipose triglyceride lipase to an anabolic treatment regime. PTH increased the release of fatty acids from adipocytes and B-oxidation by osteoblasts, while the genetic mouse models were resistant to the hormone’s anabolic effect. Collectively, these data suggest that PTH’s anabolic actions requires coordinated signaling between bone and adipose, wherein a lipolytic response liberates fatty acids that are oxidized by osteoblasts to fuel bone formation
Nathalie Alekos, Priyanka Kushwaha, Soohyun Kim, Zhu Li, Abdullah Abood, Naomi Dirckx, Susan Aja, Joe Kodama, Jean Garcia-Diaz, Satoru Otsuru, Elizabeth Rendina-Ruedy, Michael J. Wolfgang, Ryan C. Riddle
The main estrogen, estradiol (E2), exerts several beneficial vascular actions through estrogen receptor (ER)α in endothelial cells. However, the impact of other natural estrogens such as estriol (E3) and estetrol (E4) on arteries remains poorly described. In the present study, we reported the effects of E3 and E4 on endothelial healing after carotid artery injuries in vivo. After endovascular injury, that preserves smooth muscle cells (SMCs), E2, E3 and E4 equally stimulated reendothelialization. By contrast, only E2 and E3 accelerated endothelial healing after perivascular injury that destroys both endothelial cells and SMCs, suggesting an important role of this latter cell type in E4 action, which was confirmed using Cre/lox mice inactivating ERα in SMCs. In addition, E4 mediated its action independently of ERα membrane initiated signaling by contrast to E2. Consistently, RNAseq analysis revealed that transcriptomic and cellular signatures in response to E4 profoundly differ from those of E2. Thus, whereas acceleration of endothelial healing by estrogens was viewed as entirely dependent on endothelial ERα, these results highlight the very specific pharmacological profile of the natural estrogen E4, revealing the importance of dialogue between SMCs and endothelial cells in its arterial protection.
Morgane Davezac, Rana Zahreddine, Melissa Buscato, Natalia F. Smirnova, Chanaelle Febrissy, Henrik Laurell, Silveric Gilardi-Bresson, Marine Adlanmerini, Philippe Liere, Gilles Flouriot, Rachida Guennoun, Muriel Laffargue, Jean-Michel Foidart, Françoise Lenfant, Jean-François Arnal, Raphaël Métivier, Coralie Fontaine
In vertebrate species, fertility is controlled by gonadotropin-releasing hormone (GnRH) neurons. GnRH cells arise outside the central nervous system, in the developing olfactory pit, and migrate along olfactory/vomeronasal/terminal nerve axons into the forebrain during embryonic development. Congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS) are rare genetic disorders characterized by infertility and they are associated with defects in GnRH neuron migration and/or altered GnRH secretion and signaling. Here, we documented the expression of Jagged 1/Notch signaling pathway in GnRH neurons and along the GnRH neuron migratory route both in zebrafish embryos and in human fetuses. Genetic knock-down of the zebrafish ortholog of JAG1 (jag1b) resulted in altered GnRH migration and olfactory axonal projections to the olfactory bulbs. Next-generation sequencing was performed in 467 CHH unrelated probands leading to the identification of heterozygous rare variants in JAG1. Functional in vitro validation of JAG1 mutants revealed that 7 out of the 9 studied variants exhibit reduced protein levels and altered subcellular localization. Altogether our data provide compelling evidence that Jag1/Notch signaling plays a prominent role in the development of GnRH neurons and we propose that JAG1 insufficiency may contribute to the pathogenesis of CHH in humans.
Ludovica Cotellessa, Federica Marelli, Paolo Duminuco, Michela Adamo, Georgios E. Papadakis, Lucia Bartoloni, Naoko Sato, Mariarosaria Lang-Muritano, Amineh Troendle, Waljit S. Dhillo, Annamaria Morelli, Giulia Guarnieri, Nelly Pitteloud, Luca Persani, Marco Bonomi, Paolo Giacobini, Valeria Vezzoli
Dietary potassium (K+) supplementation is associated with a blood pressure (BP) lowering effect, but not all studies agree. Here we examined the effects of short and long-term K+ supplementation on BP in mice, whether differences depend on the accompanying anion or the sodium (Na+) intake and molecular alterations in the kidney that may underlie BP changes. Relative to the control diet, BP was higher in mice fed a high NaCl (1.57% Na+) for 7 weeks or 2 weeks with a K+-free diet. BP was highest on a K+-free/high NaCl diet. Commensurate with increased abundance and phosphorylation of the thiazide sensitive sodium-chloride-cotransporter (NCC) on the K+-free/high NaCl diet, BP returned to normal with thiazides. Three weeks of a high K+ diet (5% K+) increased BP (predominantly during night-time) independently of dietary Na+ or anion intake. Conversely, 4 days of KCl feeding reduced BP. Both feeding periods resulted in lower NCC levels, but increased levels of cleaved (active) α and γ subunits of the epithelial Na+ channel ENaC. The elevated BP after chronic K+ feeding was reduced by amiloride but not thiazide. Our results suggest that dietary K+ has an optimal threshold where it may be most effective for cardiovascular health.
Robert Little, Sathish K. Murali, Søren B. Poulsen, Paul R. Grimm, Adrienne Assmus, Lei Cheng, Jessica R. Ivy, Ewout J. Hoorn, Vladimir V. Matchkov, Paul A. Welling, Robert A. Fenton
Neutrophils (PMN) are the first immune responders to infection/injury playing a critical role in clearing invading microbes and promoting tissue repair. However, dysregulated trafficking of PMNs across mucosal surfaces is a pathological hallmark of numerous diseases characterized by persistent or intermittent bursts of mucosal inflammation. The critical final step in PMN trafficking into mucosal lined organs (including the lungs, kidneys, skin and gut) involves transepithelial migration (TEpM). The glycoprotein CD11b/CD18 is the predominant β2 integrin that mediates PMN TEpM. Furthermore, CD11b/CD18 also regulates key PMN inflammatory effector functions that are implicated in the pathogenesis of chronic mucosal inflammation including superoxide release and degranulation. Recent studies have shown that terminal Fucose and GlcNAc glycans on CD11b/CD18 can be targeted to reduce PMN trafficking across intestinal epithelium, highlighting the importance of glycosylation in regulating PMN inflammatory function in mucosal settings. Previous studies have also demonstrated that the most abundant terminal glycan on human and murine PMN is sialic acid (Sia). However, the role of Sia in regulating PMN epithelial influx and mucosal inflammatory function is not well understood. Here we demonstrate that inhibiting sialidase mediated removal of α2-3 linked Sia from CD11b/CD18 inhibits PMN migration across intestinal epithelium in vitro and in vivo. Sialylation was also found to regulate critical PMN inflammatory effector functions including degranulation and superoxide release. Finally, we demonstrate that sialidase inhibition reduces bacterial peptide mediated CD11b/CD18 activation in PMN and blocks downstream intracellular signaling mediated by Spleen tyrosine kinase (Syk) and p38 MAP kinase. Taken together, these data demonstrate that sialylated glycans on CD11b/CD18 represent novel targets for ameliorating PMN mediated tissue damage and reducing inflammation in mucosal inflammatory disorders.
Veronica Azcutia, Matthias Kelm, Dylan Fink, Richard D. Cummings, Asma Nusrat, Charles A. Parkos, Jennifer C. Brazil
Antisense oligonucleotide (AO)-mediated therapy is a promising strategy to treat several neurological diseases including spinal muscular atrophy (SMA). However, limited delivery to the central nervous system (CNS) with AOs administered intravenously or subcutaneously is a major challenge. Here we demonstrate a single subcutaneous administration of cell-penetrating peptide DG9 conjugated to an AO called phosphorodiamidate morpholino oligomers (PMOs) reaches the CNS and significantly prolonged the median survival compared to unconjugated PMO and R6G-PMO in a severe SMA mouse model. Treated mice exhibited significantly higher expression of full-length SMN2 expression (FL-SMN2) in both the CNS and systemic tissues compared to non-treated and unmodified AO-treated mice. The treatment ameliorated the atrophic musculature and improved breathing function accompanied by improved muscle strength and innervation at the neuromuscular junction with no signs of apparent toxicity. We also demonstrated DG9-conjugated PMO localizes in nuclei in the spinal cord and brain after subcutaneous injections. Our data identify DG9 peptide conjugation as a powerful way to improve the efficacy of AO-mediated splice modulation. Finally, DG9-PMO is a promising therapeutic option to treat SMA and other neurological diseases, overcoming the necessity for intrathecal injections and treating body-wide tissues without apparent toxicity.
Tejal Aslesh, Esra Erkut, Jun Ren, Kenji Rowel Q. Lim, Stanley Woo, Susan Hatlevig, Hong M. Moulton, Simon Gosgnach, John Greer, Rika Maruyama, Toshifumi Yokota
Vascular calcification (VC) is concomitant with atherosclerosis, yet it remains uncertain why rupture-prone high-risk plaques do not typically show extensive calcification. Intraplaque hemorrhage (IPH) deposits erythrocyte-derived cholesterol enlarging the necrotic core and promoting the high-risk plaque development. Pro-atherogenic CD163+ alternative macrophages engulf hemoglobin-haptoglobin (HH) complexes at IPH sites. However, their role in VC has never been examined. Here we show, in human arteries, the distribution of CD163+ macrophages correlates inversely with VC. In vitro experiments using vascular smooth muscle cells (VSMC) cultured with HH-exposed human macrophages supernatant (M(Hb)) reduced calcification, while arteries from ApoE-/-CD163-/- mice showed greater VC. M(Hb) supernatant-exposed VSMC showed activated NFκB, while blocking NFκB attenuated the anti-calcific effect of M(Hb) on VSMCs. CD163+ macrophages altered VC through NFκB-induced transcription of hyaluronan synthase (HAS), an enzyme which catalyzes the formation of the extracellular matrix glycosaminoglycan, hyaluronan, within VSMCs. M(Hb) supernatants enhanced HAS production in VSMC, while knocking-down HAS attenuated its anti-calcific effect. NFκB blockade in ApoE-/- mice reduced hyaluronan and increased VC. In human arteries, hyaluronan/HAS were increased in areas of CD163+ macrophage presence. Our findings highlight an important mechanism by which CD163+ macrophages inhibit VC through NFκB-induced HAS augmentation and thus promote the high-risk plaques development.
Atsushi Sakamoto, Rika Kawakami, Masayuki Mori, Liang Guo, Ka Hyun Paek, Jose Verdezoto Mosquera, Anne Cornelissen, Saikat Kumar B. Ghosh, Kenji Kawai, Takao Konishi, Raquel Fernandez, Daniela T. Fuller, Weili Xu, Aimee E. Vozenilek, Yu Sato, Hiroyuki Jinnouchi, Sho Torii, Adam W. Turner, Hirokuni Akahori, Salome Kuntz, Craig C. Weinkauf, Parker J. Lee, Robert Kutys, Kathryn Harris, Alfred Lawrence Killey, Christina M. Mayhew, Matthew Ellis, Leah M. Weinstein, Neel V. Gadhoke, Roma Dhingra, Jeremy Ullman, Endale Armelle Dikongue Emene, Maria E. Romero, Frank D. Kolodgie, Clint L. Miller, Renu Virmani, Aloke V. Finn
Reactivation of human cytomegalovirus (HCMV) from latency is a frequent complication following hematopoietic stem cell transplantation (HSCT). The development of acute graft-versus-host disease (GVHD) is a significant risk factor for HCMV disease. Using a murine GVHD model in animals latently infected with murine CMV (MCMV) we studied preventive and therapeutic interventions in this high-risk scenario of HSCT. Mice latently infected with MCMV reactivated MCMV and developed disseminated MCMV infection concomitant with the manifestations of GVHD. Dissemination was accompanied by accelerated mortality. We demonstrate that MCMV reactivation and dissemination was modulated by MCMV-specific antibodies, thus demonstrating in vivo protective activity of antiviral antibodies. However, the efficacy of serum therapy required repetitive doses of high titer immune serum secondary to the shortened serum half-life of IgG in animals with GvHD. In a complementary approach, treatment of GVHD by adoptive transfer of donor-derived regulatory T cells facilitated production of MCMV-specific antibodies from newly developing donor-derived B cells. Together, our findings strongly suggest that antibodies play a major role in controlling recurrent MCMV infection that follows GVHD and argue for reassessing the potential of antibody treatments as well as therapeutic strategies that enhance de novo antibody development against HCMV.
Martina Seefried, Nadine Hundhausen, Irena Kroeger, Maike Büttner-Herold, Petra Hoffmann, Matthias Edinger, Evelyn Ullrich, Friederike Berberich-Siebelt, William J. Britt, Michael Mach, Thomas H. Winkler
As the COVID-19 pandemic continues, long-term immunity against SARS-CoV-2 will be globally important. Official weekly cases have not dropped below 2 million since September of 2020, and continued emergence of novel variants have created a moving target for our immune systems and public health alike. The temporal aspects of COVID-19 immunity, particularly from repeated vaccination and infection, are less well understood than short-term vaccine efficacy. In this study, we explore the impact of combined vaccination and infection, also known as hybrid immunity, and the timing thereof on the quality and quantity of antibodies elicited in a cohort of 96 health care workers. We find robust neutralizing antibody responses among those with hybrid immunity against all variants, including Omicron BA.2, and significantly improved neutralizing titers with longer vaccine-infection intervals up to 400 days. These results indicate that anti-SARS-CoV-2 antibody responses undergo continual maturation following primary exposure by either vaccination or infection for at least 400 days after last antigen exposure. We show that neutralizing antibody responses improved upon secondary boosting with greater potency seen after extended intervals. Our findings may also extend to booster vaccine doses, a critical consideration in future vaccine campaign strategies.
Timothy A. Bates, Hans C. Leier, Savannah K. McBride, Devin Schoen, Zoe L. Lyski, David D. Xthona Lee, William B. Messer, Marcel E. Curlin, Fikadu G. Tafesse
Bone metastases are a common complication of breast cancer. We have demonstrated that intermittent administration of parathyroid hormone (PTH [1-34]) reduces the incidence of bone metastases in murine models of breast cancer by acting on osteoblasts to alter the bone microenvironment. Here, we examined the role of PTH receptor (PTH1R)-mediated signaling in both osteoblasts and breast cancer cells in influencing bone metastases. In mice with impaired PTH1R signaling in osteoblasts, intermittent PTH did not reduce bone metastasis. Intermittent PTH also failed to reduce bone metastasis when expression of PTH1R was knocked down in 4T1 murine breast cancer cells by shRNA. In 4T1 breast cancer cells, PTH decreased expression of PTH-related protein (PTHrP), implicated in the vicious cycle of bone metastases. Knockdown of PTHrP in 4T1 cells significantly reduced migration towards MC3T3-E1 osteoblasts, and migration was further inhibited by treatment with intermittent PTH. Conversely, overexpression of PTHrP in 4T1 cells increased migration towards MC3T3-E1 osteoblasts and this was not inhibited by PTH. In conclusion, PTH1R expression is crucial in both osteoblasts and breast cancer cells for PTH to reduce bone metastases and in breast cancer cells this may be mediated in part by suppression of PTHrP.
Srilatha Swami, Hui Zhu, Aria Nisco, Takaharu Kimura, Matthew J. Kim, Vaisakh Nair, Joy Y. Wu
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