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Individuals with chronic kidney disease have elevated levels of oxidative stress and are at a significantly higher risk of skeletal fracture. Advanced glycation end products (AGEs), which accumulate in bone and compromise mechanical properties, are known to be driven in part by oxidative stress. The goal of this study was to study effects of N-acetylcysteine (NAC) on reducing oxidative stress and improving various bone parameters, most specifically mechanical properties, in an animal model of progressive CKD. Male Cy/+ (CKD) rats and unaffected littermates were untreated (controls) or treated with NAC (80 mg/kg, IP) from 30 to 35 weeks of age. Endpoint measures included serum biochemistries, assessments of systemic oxidative stress, bone morphology, and mechanical properties, and AGE levels in the bone. CKD rats had the expected phenotype that included low kidney function, elevated parathyroid hormone, higher cortical porosity, and compromised mechanical properties. NAC treatment had mixed effects on oxidative stress markers, significantly reducing TBARS (a measure of lipid peroxidation) while not affecting 8-OHdG (a marker of DNA oxidation) levels. AGE levels in the bone were elevated in CKD animals and were reduced with NAC although this did not translate to a benefit in bone mechanical properties. In conclusion, NAC failed to significantly improve bone architecture/geometry/mechanical properties in our rat model of progressive CKD.
Fibrosis accompanying wound healing can drive the failure of many different organs. Activated fibroblasts are the principal determinants of post-injury pathological fibrosis along with physiological repair, making them a difficult therapeutic target. Although activated fibroblasts are phenotypically heterogeneous, they are not recognized as distinct functional entities. Using mice that express GFP under the FSP1 or αSMA promoter, we characterized two non-overlapping fibroblast subtypes from mouse hearts after myocardial infarction. Here, we report the identification of FSP1-GFP cells as a non-pericyte, non-hematopoietic fibroblast subpopulation with a predominant pro-angiogenic role, characterized by in vitro phenotypic/cellular/ultrastructural studies and in vivo granulation tissue formation assays combined with transcriptomics and proteomics. This work identifies a fibroblast subtype that is functionally distinct from the pro-fibrotic αSMA-expressing myofibroblast subtype. Our study has the potential to shift our focus towards viewing fibroblasts as molecularly and functionally heterogeneous and provides a paradigm to approach treatment for organ fibrosis.
Staphylococcus aureus is able to infect virtually all organ systems and is a frequently isolated etiologic agent of osteomyelitis, a common and debilitating invasive infection of bone. Treatment of osteomyelitis requires invasive surgical procedures and prolonged antibiotic therapy, yet is frequently unsuccessful due to extensive pathogen-induced bone damage that can limit antibiotic penetration and immune cell influx to the infectious focus. We previously established that S. aureus triggers profound alterations in bone remodeling in a murine model of osteomyelitis, in part through the production of osteolytic toxins. However, staphylococcal strains lacking osteolytic toxins still incite significant bone destruction, suggesting that host immune responses are also major drivers of pathologic bone remodeling during osteomyelitis. The objective of this study was to identify host immune pathways that contribute to antibacterial immunity during S. aureus osteomyelitis, and to define how these immune responses alter bone homeostasis and contribute to bone destruction. We specifically focused on the interleukin-1 receptor (IL-1R) and downstream adapter protein MyD88 given the prominent role of this signaling pathway in both antibacterial immunity and osteo-immunologic crosstalk. We discovered that while IL-1R signaling is necessary for local control of bacterial replication during osteomyelitis, it also contributes to bone loss during infection. Mechanistically, we demonstrate that S. aureus enhances osteoclastogenesis of myeloid precursors in vitro, and increases the abundance of osteoclasts residing on bone surfaces in vivo. This enhanced osteoclast abundance translates to trabecular bone loss, and is dependent on intact IL-1R signaling. Collectively, these data define IL-1R signaling as a critical component of the host response to S. aureus osteomyelitis, but also demonstrate that IL-1R-dependent immune responses trigger collateral bone damage through activation of osteoclast-mediated bone resorption.
Hematopoiesis is a dynamic system that requires balanced cell division, differentiation, and death. The 2 major modes of programmed cell death, apoptosis and necroptosis, share molecular machinery but diverge in outcome with important implications for the microenvironment; apoptotic cells are removed in an immune silent process, whereas necroptotic cells leak cellular contents that incite inflammation. Given the importance of cytokine-directed cues for hematopoietic cell survival and differentiation, the impact on hematopoietic homeostasis of biasing cell death fate to necroptosis is substantial and poorly understood. Here, we present a mouse model with increased bone marrow necroptosis. Deletion of the proapoptotic Bcl-2 family members Bax and Bak inhibits bone marrow apoptosis. Further deletion of the BH3-only member Bid (to generate triple-knockout [TKO] mice) leads to unrestrained bone marrow necroptosis driven by increased Rip1 kinase (Ripk1). TKO mice display loss of progenitor cells, leading to increased cytokine production and increased stem cell proliferation and exhaustion and culminating in bone marrow failure. Genetically restoring Ripk1 to wild-type levels restores peripheral red cell counts as well as normal cytokine production. TKO bone marrow is hypercellular with abnormal differentiation, resembling the human disorder myelodysplastic syndrome (MDS), and we demonstrate increased necroptosis in MDS bone marrow. Finally, we show that Bid impacts necroptotic signaling through modulation of caspase-8-mediated Ripk1 degradation. Thus, we demonstrate that dysregulated necroptosis in hematopoiesis promotes bone marrow progenitor cell death that incites inflammation, impairs hematopoietic stem cells, and recapitulates the salient features of the bone marrow failure disorder MDS.
© 2019 by The American Society of Hematology.
IMPACT STATEMENT - The development of autograft extenders is a significant clinical need in bone tissue engineering. We report new settable poly(thioketal urethane)-based autograft extenders that have bone-like mechanical properties and handling properties comparable to calcium phosphate bone cements. These settable autograft extenders remodeled to form new bone in a biologically stringent intertransverse process model of bone formation that does not heal when treated with calcium phosphate bone void fillers or cements alone. This is the first study to report settable autograft extenders with bone-like strength and handling properties comparable to ceramic bone cements, which have the potential to improve treatment of bone fractures and other orthopedic conditions.
Greater understanding of the determinants of skeletal fragility is highly sought due to the great burden that bone affecting diseases and fractures have on economies, societies and health care systems. Being a complex, hierarchical composite of collagen type-I and non-stoichiometric substituted hydroxyapatite, bone derives toughness from its organic phase. In this study, we tested whether early observations that a strong correlation between bone collagen integrity measured by thermomechanical methods and work to fracture exist in a more general and heterogeneous sampling of the population. Neighboring uniform specimens from an established, highly characterized and previously published collection of human cortical bone samples (femur mid-shaft) were decalcified in EDTA. Fifty-four of the original 62 donors were included (26 male and 28 females; ages 21-101 years; aging, osteoporosis, diabetes and cancer). Following decalcification, bone collagen was tested using hydrothermal isometric tension (HIT) testing in order to measure the collagen's thermal stability (denaturation temperature, T) and network connectivity (maximum rate of isometric tension generation; Max.Slope). We used linear regression and general linear models (GLMs) with several explanatory variables to determine whether relationships between HIT parameters and generally accepted bone quality factors (e.g., cortical porosity, pentosidine content [pen], pyridinoline content [pyd]), age, and measures of fracture toughness (crack initiation fracture toughness, K, and total energy release/dissipation rate evaluated at the point of unstable fast fracture, J-int) were significant. Bone collagen connectivity (Max.Slope) correlated well with the measures of fracture toughness (R = 24-35%), and to a lesser degree with bound water fraction (BW; R = 7.9%) and pore water fraction (PW; R = 9.1%). Significant correlations with age, apparent volumetric bone mineral density (vBMD), and mature enzymatic [pyd] and non-enzymatic collagen crosslinks [pen] were not detected. GLMs found that Max.Slope and vBMD (or BW), with or without age as additional covariate, all significantly explained the variance in Kinit (adjusted-R = 36.7-49.0%). Also, the best-fit model for J-int (adjusted-R = 35.7%) included only age and Max.Slope as explanatory variables with Max.Slope contributing twice as much as age. Max.Slope and BW without age were also significant predictors of J-int (adjusted-R = 35.5%). In conclusion, bone collagen integrity as measured by thermomechanical methods is a key factor in cortical bone fracture toughness. This study further demonstrates that greater attention should be paid to degradation of the overall organic phase, rather than a specific biomarker (e.g. [pen]), when seeking to understand elevated fracture rates in aging and disease.
Copyright © 2018 Elsevier Inc. All rights reserved.
TGF-β signals through a receptor complex composed of 2 type I and 2 type II (TGF-βRII) subunits. We investigated the role of macrophage TGF-β signaling in fibrosis after AKI in mice with selective monocyte/macrophage TGF-βRII deletion (macrophage TGF-βRII-/- mice). Four weeks after injury, renal TGF-β1 expression and fibrosis were higher in WT mice than macrophage TGF-βRII-/- mice, which had decreased renal macrophages. The in vitro chemotactic response to f-Met-Leu-Phe was comparable between bone marrow-derived monocytes (BMMs) from WT and macrophage TGF-βRII-/- mice, but TGF-βRII-/- BMMs did not respond to TGF-β. We then implanted Matrigel plugs suffused with either f-Met-Leu-Phe or TGF-β1 into WT or macrophage TGF-βRII-/- mice. After 6 days, f-Met-Leu-Phe induced similar macrophage infiltration into the Matrigel plugs of WT and macrophage TGF-βRII-/- mice, but TGF-β induced infiltration only in WT mice. We further determined the number of labeled WT or TGF-βRII-/- BMMs infiltrating into WT kidneys 20 days after ischemic injury. There were more labeled WT BMMs than TGF-βRII-/- BMMs. Therefore, macrophage TGF-βRII deletion protects against the development of tubulointerstitial fibrosis following severe ischemic renal injury. Chemoattraction of macrophages to the injured kidney through a TGF-β/TGF-βRII axis is a heretofore undescribed mechanism by which TGF-β can mediate renal fibrosis during progressive renal injury.
We present an integromic analysis of gene alterations that modulate transforming growth factor β (TGF-β)-Smad-mediated signaling in 9,125 tumor samples across 33 cancer types in The Cancer Genome Atlas (TCGA). Focusing on genes that encode mediators and regulators of TGF-β signaling, we found at least one genomic alteration (mutation, homozygous deletion, or amplification) in 39% of samples, with highest frequencies in gastrointestinal cancers. We identified mutation hotspots in genes that encode TGF-β ligands (BMP5), receptors (TGFBR2, AVCR2A, and BMPR2), and Smads (SMAD2 and SMAD4). Alterations in the TGF-β superfamily correlated positively with expression of metastasis-associated genes and with decreased survival. Correlation analyses showed the contributions of mutation, amplification, deletion, DNA methylation, and miRNA expression to transcriptional activity of TGF-β signaling in each cancer type. This study provides a broad molecular perspective relevant for future functional and therapeutic studies of the diverse cancer pathways mediated by the TGF-β superfamily.
Copyright © 2018 Elsevier Inc. All rights reserved.
At least 26% of recent battlefield injuries are to the craniomaxillofacial (CMF) region. Recombinant human bone morphogenetic protein 2 (rhBMP-2) is used to treat CMF open fractures, but several complications have been associated with its use. This study tested the efficacy and safety of a lower (30% recommended) dose of rhBMP-2 to treat mandibular fractures. rhBMP-2 delivered via a polyurethane (PUR) and hydroxyapatite/β-tricalcium phosphate (Mastergraft®) scaffold was evaluated in a 2 cm segmental mandibular defect in minipigs. Bone regeneration was analyzed at 4, 8, and 12 weeks postsurgery using clinical computed tomography (CT) and rhBMP-2, and inflammatory marker concentrations were analyzed in serum and surgery-site drain effluent. CT scans revealed that pigs treated with PUR-Mastergraft® + rhBMP-2 had complete bone bridging, while the negative control group showed incomplete bone-bridging (n = 6). Volumetric analysis of regenerated bone showed that the PUR-Mastergraft® + rhBMP-2 treatment generated significantly more bone than control by 4 weeks, a trend that continued through 12 weeks. Variations in inflammatory analytes were detected in drain effluent samples and saliva but not in serum, suggesting a localized healing response. Importantly, the rhBMP-2 group did not exhibit an excessive increase in inflammatory analytes compared to control. Treatment with low-dose rhBMP-2 increases bone regeneration capacity in pigs with mandibular continuity defects and restores bone quality. Negative complications from rhBMP-2, such as excessive inflammatory analyte levels, were not observed. Together, these results suggest that treatment with low-dose rhBMP-2 is efficacious and may improve safety when treating CMF open fractures. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1491-1503, 2019.
© 2018 Wiley Periodicals, Inc.