Alteration of blood clot structures by interleukin-1 beta in association with bone defects healing

The quality of hematomas are crucial for successful early bone defect healing, as the structure of fibrin clots can significantly influence the infiltration of cells, necessary for bone regeneration, from adjacent tissues into the fibrin network. This study investigated if there were structural differences between hematomas from normal and delayed healing bone defects and whether such differences were linked to changes in the expression of IL-1β. Using a bone defect model in rats, we found that the hematomas in the delayed healing model had thinner fibers and denser clot structures. Moreover, IL-1β protein levels were significantly higher in the delayed healing hematomas. The effects of IL-1β on the structural properties of human whole blood clots were evaluated by thrombelastograph (TEG), scanning electronic microscopy (SEM), compressive study, and thrombolytic assays. S-nitrosoglutathione (GSNO) was applied to modulate de novo hematoma structure and the impact on bone healing was evaluated in the delayed healing model. We found that GSNO produced more porous hematomas with thicker fibers and resulted in significantly enhanced bone healing. This study demonstrated that IL-1β and GSNO had opposing effects on clot architecture, the structure of which plays a pivotal role in early bone healing

Macrophage apolipoprotein-E knockdown modulates caspase-3 activation without altering sensitivity to apoptosis

Apolipoprotein-E (apoE) expression may be associated with apoptosis resistance. Since macrophages constitutively synthesize apoE we speculated that this may contribute to apoptosis resistance. Using siRNA, human monocyte derived macrophage (hMDM) apoE mRNA and protein was reduced by 97% and 61%, respectively. ApoE knockdown increased staurosporine-induced caspase-3 activation by 78% without altering cell survival or apoptosis as assessed by TUNEL analysis and morphological changes. This result was confirmed using murine bone marrow derived macrophages (mBMDM) from apoE null and wild type mice. In these experiments, staurosporine-induced caspase-3 activation was increased by 49% in apoE null compared to wild type mBMDM and this was not associated with differences in TUNEL signal, annexin-V binding or DNA fragmentation. ApoE is also important for cholesterol transport and macrophage cholesterol can regulate apoptosis. Knockdown of hMDM apoE inhibited basal cholesterol efflux by 20% without altering apolipoprotein-AI mediated cholesterol efflux over 24 h. Similarly, in apoE null mBMDM a non significant trend for a 16% reduction in basal cholesterol efflux was observed as compared to wild type mBMDM. In conclusion, apoE expression modulates capase-3 activity, but this has no significant impact on sensitivity to apoptosis and only a moderate impact on basal cholesterol efflux. © 2007 Elsevier B.V. All rights reserved