Modelling neurofibromatosis type 1 tibial dysplasia and its treatment with lovastatin

<p>Abstract</p> <p>Background</p> <p>Bowing and/or pseudarthrosis of the tibia is a known severe complication of neurofibromatosis type 1 (NF1). Mice with conditionally inactivated neurofibromin (Nf1) in the developing limbs and cranium (Nf1Prx1) show bowing of the tibia caused by decreased bone mineralisation and increased bone vascularisation. However, in contrast to NF1 patients, spontaneous fractures do not occur in Nf1Prx1 mice probably due to the relatively low mechanical load. We studied bone healing in a cortical bone injury model in Nf1Prx1 mice as a model for NF1-associated bone disease. Taking advantage of this experimental model we explore effects of systemically applied lovastatin, a cholesterol-lowering drug, on the Nf1 deficient bone repair.</p> <p>Methods</p> <p>Cortical injury was induced bilaterally in the <it>tuberositas tibiae </it>in Nf1Prx1 mutant mice and littermate controls according to a method described previously. Paraffin as well as methacrylate sections were analysed from each animal. We divided 24 sex-matched mutant mice into a lovastatin-treated and an untreated group. The lovastatin-treated mice received 0.15 mg activated lovastatin by daily gavage. The bone repair process was analysed at three consecutive time points post injury, using histological methods, micro computed tomography measurements and <it>in situ </it>hybridisation. At each experimental time point, three lovastatin-treated mutant mice, three untreated mutant mice and three untreated control mice were analysed. The animal group humanely killed on day 14 post injury was expanded to six treated and six untreated mutant mice as well as six control mice.</p> <p>Results</p> <p>Bone injury repair is a complex process, which requires the concerted effort of numerous cell types. It is initiated by an inflammatory response, which stimulates fibroblasts from the surrounding connective tissue to proliferate and fill in the injury site with a provisional extracellular matrix. In parallel, mesenchymal progenitor cells from the periost are recruited into the injury site to become osteoblasts. In Nf1Prx1 mice bone repair is delayed and characterised by the excessive formation and the persistence of fibro-cartilaginous tissue and impaired extracellular matrix mineralisation. Correspondingly, expression of Runx2 is downregulated. High-dose systemic lovastatin treatment restores Runx2 expression and accelerates new bone formation, thus improving cortical bone repair in Nf1Prx1 tibia. The bone anabolic effects correlate with a reduction of the mitogen activated protein kinase pathway hyper-activation in Nf1-deficient cells.</p> <p>Conclusion</p> <p>Our data suggest the potential usefulness of lovastatin, a drug approved by the US Food and Drug Administration in 1987 for the treatment of hypercholesteraemia, in the treatment of Nf1-related fracture healing abnormalities. The experimental model presented here constitutes a valuable tool for the pre-clinical stage testing of candidate drugs, targeting Nf1-associated bone dysplasia.</p

Twenty-three unsolved problems in hydrology (UPH) – a community perspective

This paper is the outcome of a community initiative to identify major unsolved scientific problems in hydrology motivated by a need for stronger harmonisation of research efforts. The procedure involved a public consultation through online media, followed by two workshops through which a large number of potential science questions were collated, prioritised, and synthesised. In spite of the diversity of the participants (230 scientists in total), the process revealed much about community priorities and the state of our science: a preference for continuity in research questions rather than radical departures or redirections from past and current work. Questions remain focused on the process-based understanding of hydrological variability and causality at all space and time scales. Increased attention to environmental change drives a new emphasis on understanding how change propagates across interfaces within the hydrological system and across disciplinary boundaries. In particular, the expansion of the human footprint raises a new set of questions related to human interactions with nature and water cycle feedbacks in the context of complex water management problems. We hope that this reflection and synthesis of the 23 unsolved problems in hydrology will help guide research efforts for some years to come.publishedVersio

Loss of murine Gfi1 causes neutropenia and induces osteoporosis depending on the pathogen load and systemic inflammation.

Gfi1 is a key molecule in hematopoietic lineage development and mutations in GFI1 cause severe congenital neutropenia (SCN). Neutropenia is associated with low bone mass, but the underlying mechanisms are poorly characterized. Using Gfi1 knock-out mice (Gfi1-ko/ko) as SCN model, we studied the relationship between neutropenia and bone mass upon different pathogen load conditions. Our analysis reveals that Gfi1-ko/ko mice kept under strict specific pathogen free (SPF) conditions demonstrate normal bone mass and survival. However, Gfi1-ko/ko mice with early (nonSPF) or late (SPF+nonSPF) pathogen exposure develop low bone mass. Gfi1-ko/ko mice demonstrate a striking rise of systemic inflammatory markers according to elevated pathogen exposure and reduced bone mass. Elevated inflammatory cytokines include for instance Il-1b, Il-6, and Tnf-alpha that regulate osteoclast development. We conclude that low bone mass, due to low neutrophil counts, is caused by the degree of systemic inflammation promoting osteoclastogenesis

Differential bone marrow cell count of Gfi1 mice kept under SPF and SPF+nonSPF conditions.

<p>Differential bone marrow cell count of Gfi1 mice kept under SPF and SPF+nonSPF conditions.</p

Gfi1 regulates hematopoietic and mesenchymal cells. Low bone mass in Gfi1-ko/ko mice results from inflammatory response due to severe neutropenia.

<p>Loss of Gfi1 and housing at conditions of variable pathogen load lead to increased mortality, inflammatory response, and reduction of bone mass. The inflammatory response is the main determinant of osteopenia and osteoporosis in the severe neutropenia mouse model Gfi1.</p

Gfi1-ko/ko mutants upon SPF+nonSPF housing show elevated osteoclast activity and bone cell marker expression.

<p><b>(A)</b> In Gfi1-ko/ko mutant mice kept under SPF and SPF+nonSPF Rankl plasma levels are unaffected and Opg is elevated compared to Gfi1-wt/wt mice. Bone resorption (CTX-I) is mildly increased in Gfi1-ko/ko mice under both conditions. Bone formation (PINP) is mildly reduced in SPF Gfi1-ko/ko mice compared to Gfi1-wt/wt but not under SPF+nonSPF conditions. Error bars represent SD and statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01. <b>(B)</b> Relative expression of bone marker mRNA was analyzed with qPCR and Gapdh was used as endogenous control. Results are presented as ratio Gfi1-ko/ko vs. Gfi1-wt/wt (n = 3 with 3 technical replicates/sample; error bar indicates SD). Normal expression is indicated with the dotted line at 1. All Ob. specific markers such as Col1a1, Runx2, and Spp1 are approx. 2-fold elevated. <b>(C)</b> Markers indicating mature Oc. such as Acp5 and Ctsk are approx. 4-fold increased. Interestingly, the marker for Oc. progenitor cells Sfip1 (Pu.1) is also 4-fold elevated. Relative expression of Oc. marker mRNA was analyzed with qPCR and Gapdh was used as endogenous control.</p

Gfi1-ko/ko mice kept under nonSPF conditions develop osteopenia.

<p><b>(A)</b> Trabecular bone of vertebrae was measured with microCT. Gfi1-ko/ko mice kept under nonSPF conditions developed severe osteopenia as indicated by 60% BV/TV reduction compared to Gfi1-wt/wt mice. Upon SPF breeding Gfi1-ko/ko mice show a 15% reduction in BV/TV. However, SPF+nonSPF conditions induced intermediate osteopenia characterized by approx. 30% BV/TV reduction. Please note Gfi1-ko/ko mice kept at nonSPF conditions develop cortical bone osteopenia in femur. <b>(B)</b> Representative bone sections stained with von Kossa/ Kernechtrot illustrate trabecular bone in vertebrae of mice kept under nonSPF, SPF and SPF+nonSPF conditions. <b>(C)</b> Quantification of bone tissue by histomorphometry revealed reduced BV/TV values in Gfi1-ko/ko vertebrae under nonSPF and SPF+nonSPF conditions compared to controls. Breeding within the SPF environment did not significantly affect bone mass of Gfi1-ko/ko mice. <b>(D)</b> Histomorphometric quantification of the osteoblast covered bone surface (Ob.S/BS) shows reduced but elevated values upon nonSPF and SPF+nonSPF breeding, respectively. SPF breeding did not affect Ob.S/BS between control and mutant mice. <b>(E)</b> Quantification of the osteoclast covered bone surface (Oc.S/BS) revealed diminished but raised values in Gfi1-ko/ko mice upon nonSPF and SPF+nonSPF breeding compared to their corresponding Gfi1-wt/wt controls, respectively. Gfi1-ko/ko mutants grown under SPF conditions showed elevated Oc.S/BS counts compared to Gfi1-wt/wt mice. For better comparability, Gfi1-wt/wt values were set to 1 and Gfi1-ko/ko values were relatively calculated for each breeding condition. Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s008" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s009" target="_blank">S3</a> Tables for absolute values. Error bars represent SD and statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01.</p

Housing conditions determine Gfi1-ko/ko mice body mass and survival.

<p><b>(A)</b> Average growth curves from control mice kept under nonSPF conditions indicate evolving development also beyond weaning (Gfi1-wt/wt n = 4, Gfi1-ko/ko n = 3). However, soon after weaning (P19) Gfi1-ko/ko mutants display significantly delayed growth. Development under SPF growth conditions did not affect growth of Gfi1-ko/ko mice (Gfi1-wt/wt n = 4, Gfi1-ko/ko n = 3). Upon SPF+nonSPF housing Gfi1-ko/ko mice demonstrated relative normal growth compared to controls (Gfi1-wt/wt n = 4, Gfi1-ko/ko n = 3); however, Gfi1-ko/ko mice show significant body mass reduction. All curves show values of male mice. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s007" target="_blank">S1 Table</a> for health monitoring. <b>(B)</b> At SPF and SPF+nonSPF conditions Gfi1-ko/ko mice showed a mortality rate of approx. 9% and 6%, respectively. <b>(C)</b> Final body mass of controls and Gfi1-ko/ko mice was assessed at indicated time points for SPF and SPF+nonSPF conditions. Combined SPF+nonSPF breeding caused a body mass reduction of approx. 25% in male and female Gfi1-ko/ko mice. Error bars represent SD and statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01.</p

Elevated G-CSF and GM-SCF but normal M-CSF levels in Gfi1-ko/ko mice.

<p>The plasma levels of M-CSF, G-CSF, and GM-CSF were assessed by ELISA in mice kept at nonSPF, SPF, and SPF+nonSPF conditions. <b>(A)</b> M-CSF levels of Gfi1-ko/ko mutants were unaffected compared to controls upon all breeding conditions. <b>(B)</b> G-CSF is massively elevated in Gfi1-ko/ko mice compared to controls housed at nonSPF, SPF, and SPF+nonSPF conditions. <b>(C)</b> At all conditions Gfi1-ko/ko mice demonstrate significantly elevated GM-CSF levels. GM-CSF levels were measured by Q-Plex ELISA assay. The GM-CSF was below the limit of detection (b.l.d.) in Gfi1-wt/wt under all breeding conditions. Error bars represent SD. <b>(D)</b> The plasma levels of inflammatory cytokines were assessed by Q-Plex assay in control and Gfi1-ko/ko mice kept at nonSPF, SPF, and SPF+nonSPF conditions. Inflammatory cytokines such as Il-1β, Il-6, IFN-gamma, and TNF-alpha are significantly elevated in Gfi1-ko/ko mice kept at SPF+nonSPF conditions compared to their controls. Highest levels of Il-1β, Il-6, IFN-gamma, and TNF-alpha are present in Gfi1-ko/ko mice exclusively kept at nonSPF conditions. Due to sample limitations we measured for nonSPF mice serum pools (n = 2 with n = 4/pool) that were not statistically evaluated. Please note that lowest cytokine concentrations in Gfi1-ko/ko mice are present upon SPF conditions. In control mice IFN-gamma was below the limit of detection independent from the breeding condition. Full data set of analyzed cytokines is available as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s011" target="_blank">S5 Table</a>. Error bars represent SEM. Statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01.</p