Cell surface heparan sulfate proteoglycans (HSPGs) have been implicated in bone morphogenetic protein (BMP)-mediated morphogenesis by regulating BMP activity and gradient formation. However, the ...direct role of HSPGs in BMP signaling is poorly understood. Here we show that HSPGs directly regulate BMP2-mediated transdifferentiation of C2C12 myoblasts into osteoblasts. HSPGs sequester BMP2 at the cell surface and mediate BMP2 internalization. Depletion of cell surface HSPGs by heparinase III treatment or decreased glycosaminoglycan chain sulfation with sodium chlorate enhances BMP2 morpho-genetic bioactivity. The addition of exogenous heparin, a widely used anticoagulant, reduced BMP2 signaling. Our results suggest that cell surface HSPGs mediate BMP2 internalization and modulate BMP2 osteogenic activity.
Cells with osteogenic potential can be found in a variety of tissues. Here we show that circulating osteogenic precursor (COP) cells, a bone marrow‐derived type I collagen+/CD45+ subpopulation of ...mononuclear adherent cells, are present in early preosseous fibroproliferative lesions in patients with fibrodysplasia ossificans progressiva (FOP) and nucleate heterotopic ossification (HO) in a murine in vivo implantation assay. Blood samples from patients with FOP with active episodes of HO contain significantly higher numbers of clonally derived COP cell colonies than patients with stable disease or unaffected individuals. The highest level of COP cells was found in a patient just before the clinical onset of an HO exacerbation. Our studies show that even COP cells derived from an unaffected individual can contribute to HO in genetically susceptible host tissue. The possibility that circulating, hematopoietic‐derived cells with osteogenic potential can seed inflammatory sites has tremendous implications and, to our knowledge, represents the first example of their involvement in clinical HO. Thus, bone formation is not limited to cells of the mesenchymal lineage, and circulating cells of hematopoietic origin can also serve as osteogenic precursors at remote sites of tissue inflammation. STEM CELLS 2009;27:2209–2219
The studies reported here were performed as part of a program in space radiation biology in which proton radiation like that present in solar particle events, as well as conventional gamma radiation, ...were being evaluated in terms of the ability to affect hemostasis.
Ferrets were exposed to 0 to 2 Gy of whole-body proton or gamma radiation and monitored for 30 days. Blood was analyzed for blood cell counts, platelet clumping, thromboelastometry, and fibrin clot formation.
The lethal dose of radiation to 50% of the population (LD50) of the ferrets was established at ∼ 1.5 Gy, with 100% mortality at 2 Gy. Hypocoagulability was present as early as day 7 postirradiation, with animals unable to generate a stable clot and exhibiting signs of platelet aggregation, thrombocytopenia, and fibrin clots in blood vessels of organs. Platelet counts were at normal levels during the early time points postirradiation when coagulopathies were present and becoming progressively more severe; platelet counts were greatly reduced at the time of the white blood cell nadir of 13 days.
Data presented here provide evidence that death at the LD50 in ferrets is most likely due to disseminated intravascular coagulation (DIC). These data question the current hypothesis that death at relatively low doses of radiation is due solely to the cell-killing effects of hematopoietic cells. The recognition that radiation-induced DIC is the most likely mechanism of death in ferrets raises the question of whether DIC is a contributing mechanism to radiation-induced death at relatively low doses in large mammals.
Interactions between transmembrane (TM) helices play an important role in the regulation of diverse biological functions. For example, the TM helices of integrins are believed to interact ...heteromerically in the resting state; disruption of this interaction results in integrin activation and cellular adhesion. However, it has been difficult to demonstrate the specificity and affinity of the interaction between integrin TM helices and to relate them to the activation process. To examine integrin TM helix associations, we developed a bacterial reporter system and used it to define the sequence motif required for helix-helix interactions in the β ₁ and β ₃ integrin subfamilies. The helices interact in a novel three-dimensional motif, the "reciprocating large-small motif" that is also observed in the crystal structures of unrelated proteins. Modest but specific stabilization of helix associations is realized via packing of complementary small and large groups on neighboring helices. Mutations destabilizing this motif activate native, full-length integrins. Thus, this highly conserved dissociable motif plays a vital and widespread role as an on-off switch that can integrate with other control elements during integrin activation.
The study of FOP, a disabling genetic disorder of progressive heterotopic ossification, is hampered by the lack of readily available connective tissue progenitor cells. We isolated such cells from ...discarded primary teeth of patients with FOP and controls and discovered dysregulation of BMP signaling and rapid osteoblast differentiation in FOP cells compared with control cells.
Introduction: Fibrodysplasia ossificans progressiva (FOP), the most disabling condition of progressive heterotopic ossification in humans, is caused by a recurrent heterozygous missense mutation in activin receptor IA (ACVR1), a bone morphogenetic protein (BMP) type I receptor, in all classically affected individuals. A comprehensive understanding of FOP has been limited, in part, by a lack of readily available connective tissue progenitor cells in which to study the molecular pathology of this disorder.
Materials and Methods: We derived connective tissue progenitor cells from discarded primary teeth (SHED cells) of patients with FOP and controls and examined BMP signaling and osteogenic differentiation in these cells.
Results: SHED cells transmitted BMP signals through both the SMAD and p38 mitogen‐activated protein kinase (MAPK) pathways and responded to BMP4 treatment by inducing BMP responsive genes. FOP cells showed ligand‐independent BMP signaling and ligand‐dependent hyper‐responsiveness to BMP stimulation. Furthermore, FOP cells showed more rapid differentiation to an osteogenic phenotype than control cells.
Conclusions: This is the first study of BMP signaling and osteogenic differentiation in connective tissue progenitor cells from patients with FOP. Our data strongly support both basal and ligand‐stimulated dysregulation of BMP signaling consistent with in silico studies of the mutant ACVR1 receptor in this condition. This study substantially extends our understanding of dysregulated BMP signaling in a progenitor cell population relevant to the pathogenesis of this catastrophic disorder of progressive ectopic ossification.
Colorimetric staining techniques such as immunohistochemistry (IHC), immunofluorescence (IF) and histochemistry (HC) provide useful information regarding the localization and relative amount of a ...molecule/substance in skin. We have developed a novel, straightforward method to assess colorimetric staining by combining features from two open‐source software programs. As a proof of principle, we demonstrate the utility of this approach by analysing changes in skin melanin deposition during the radiation‐induced tanning response of Yucatan mini‐pigs. This method includes a visualization step to validate the accuracy of colour selection before quantitation to ensure accuracy. The data show that this method is robust and will provide a means to obtain accurate comparative analyses of staining in IHC/IF/HC samples.
The temporomandibular joint (TMJ) is a diarthrodial joint that relies on lubricants for frictionless movement and long-term function. It remains unclear what temporal and causal relationships may ...exist between compromised lubrication and onset and progression of TMJ disease. Here we report that Proteoglycan 4 (Prg4)-null TMJs exhibit irreversible osteoarthritis-like changes over time and are linked to formation of ectopic mineralized tissues and osteophytes in articular disc, mandibular condyle and glenoid fossa. In the presumptive layer of mutant glenoid fossa's articulating surface, numerous chondrogenic cells and/or chondrocytes emerged ectopically within the type I collagen-expressing cell population, underwent endochondral bone formation accompanied by enhanced Ihh expression, became entrapped into temporal bone mineralized matrix, and thereby elicited excessive chondroid bone formation. As the osteophytes grew, the roof of the glenoid fossa/eminence became significantly thicker and flatter, resulting in loss of its characteristic concave shape for accommodation of condyle and disc. Concurrently, the condyles became flatter and larger and exhibited ectopic bone along their neck, likely supporting the enlarged condylar heads. Articular discs lost their concave configuration, and ectopic cartilage developed and articulated with osteophytes. In glenoid fossa cells in culture, hedgehog signaling stimulated chondrocyte maturation and mineralization including alkaline phosphatase, while treatment with hedgehog inhibitor HhAntag prevented such maturation process. In sum, our data indicate that Prg4 is needed for TMJ integrity and long-term postnatal function. In its absence, progenitor cells near presumptive articular layer and disc undergo ectopic chondrogenesis and generate ectopic cartilage, possibly driven by aberrant activation of Hh signaling. The data suggest also that the Prg4-null mice represent a useful model to study TMJ osteoarthritis-like degeneration and clarify its pathogenesis.
•Prg4-null TMJs exhibit irreversible osteoarthritis-like changes.•Osteophyte formation in Prg4-null TMJs is associated with ectopic hedgehog signaling.•Loss of Prg4 function results in abnormal endochondral bone formation in the glenoid fossa.•Prg4-mediated boundary lubrication is indispensable for TMJ maintenance.
There is a grave need for safer antiplatelet therapeutics to prevent heart attack and stroke. Agents targeting the interaction of platelets with the diseased vessel wall could impact vascular disease ...with minimal effects on normal hemostasis. We targeted integrin α₂β₁, a collagen receptor, because its overexpression is associated with pathological clot formation whereas its absence does not cause severe bleeding. Structure-activity studies led to highly potent and selective small-molecule inhibitors. Responses of integrin α₂β₁ mutants to these compounds are consistent with a computational model of their mode of inhibition and shed light on the activation mechanism of I-domain-containing integrins. A potent compound was proven efficacious in an animal model of arterial thrombosis, which demonstrates in vivo efficacy for inhibition of this platelet receptor. These results suggest that targeting integrin α₂β₁ could be a potentially safe, effective approach to long-term therapy for cardiovascular disease.
Pyogenic granuloma of the larynx: A rare cause of hemoptysis Hanick, Andrea L.; Meleca, Joseph B.; Billings, Steven D. ...
American journal of otolaryngology,
March-April 2019, 2019 Mar - Apr, 2019-03-00, 20190301, Volume:
40, Issue:
2
Journal Article
Peer reviewed
Pyogenic granuloma (PG) may involve gingival mucosa (granuloma gravidarum) in pregnancy but rarely involves the airway. This case report is perhaps the only reported presentation of PG in the larynx ...causing hemoptysis at a late stage of pregnancy. On laryngoscopic exam, a vascular, right false vocal fold neoplasm was identified with pathological characteristics consistent with PG. Conclusions: Pyogenic granuloma is a relatively common tumor of pregnancy but rarely involves the larynx. In the case of airway involvement during pregnancy, it is best managed in coordination with the high-risk obstetrical team and can be removed safely via standard microsurgical techniques.
FOP is a disorder in which skeletal muscle is progressively replaced with bone. FOP lymphocytes, a model system for exploring the BMP pathway in these patients, exhibit a defect in BMPRIA ...internalization and increased activation of downstream signaling, suggesting that altered BMP receptor trafficking underlies ectopic bone formation in this disease.
Introduction: Fibrodysplasia ossificans progressiva (FOP) is a severely disabling disorder characterized by progressive heterotopic ossification of connective tissues. Whereas the genetic defect and pathophysiology of this condition remain enigmatic, BMP4 mRNA and protein are overexpressed, and mRNAs for a subset of secreted BMP antagonists are not synthesized at appropriate levels in cultured lymphocytes from FOP patients. These data suggest involvement of altered BMP signaling in the disease. In this study, we investigate whether the abnormality is associated with defective BMP receptor function in lymphocytes.
Materials and Methods: Cell surface proteins were quantified by fluorescence‐activated cell sorting (FACS). Protein phosphorylation was assayed by immunoprecipitation and immunoblotting. Protein synthesis and degradation were examined by 35Smethionine labeling and pulse‐chase assays. mRNA was detected by RT‐PCR.
Results: FOP lymphocytes expressed 6‐fold higher levels of BMP receptor type IA (BMPRIA) on the cell surface compared with control cells and displayed a marked reduction in ligand‐stimulated internalization and degradation of BMPRIA. Moreover, in control cells, BMP4 treatment increased BMPRIA phosphorylation, whereas BMPRIA showed ligand‐insensitive constitutive phosphorylation in FOP cells. Our data additionally support that the p38 mitogen‐activated protein kinase (MAPK) signaling pathway is a major BMP signaling pathway in these cell lines and that expression of inhibitor of DNA binding and differentiation 1 (ID‐1), a transcriptional target of BMP signaling, is enhanced in FOP cells.
Conclusions: These data extend our previous observations of misregulated BMP4 signaling in FOP lymphocytes and show that cell surface overabundance and constitutive phosphorylation of BMPRIA are associated with a defect in receptor internalization. Altered BMP receptor trafficking may play a significant role in FOP pathogenesis.
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