Elevated adipogenesis of marrow mesenchymal stem cells during early steroid-associated osteonecrosis development

<p>Abstract</p> <p>Background</p> <p>Increased bone marrow lipid deposition in steroid-associated osteonecrosis (ON) implies that abnormalities in fat metabolism play an important role in ON development. The increase in lipid deposition might be explained by elevated adipogenesis of marrow mesenchymal stem cells (MSCs). However, it remains unclear whether there is a close association between elevated adipogenesis and steroid-associated ON development.</p> <p>Objective</p> <p>The present study was designed to test the hypothesis that there might be a close association between elevated adipogenesis and steroid-associated ON development.</p> <p>Methods</p> <p>ON rabbit model was induced based on our established protocol. Dynamic-MRI was employed for local intra-osseous perfusion evaluation in bilateral femora. Two weeks after induction, bone marrow was harvested for evaluating the ability of adipogenic differentiation of marrow MSCs at both cellular and mRNA level involving adipogenesis-related gene peroxisome proliferator-activated receptor gamma2 (PPARγ2). The bilateral femora were dissected for examining marrow lipid deposition by quantifying fat cell number, fat cell size, lipid deposition area and ON lesions. For investigating association among adipogenesis, lipid deposition and perfusion function with regard to ON occurrence, the rabbits were divided into ON⁺(with at least one ON lesion) group and ON^-(without ON lesion) group. For investigating association among adipogenesis, lipid deposition and perfusion function with regard to ON extension, the ON⁺rabbits were further divided into sub-single-lesion group (SON group: with one ON lesion) and sub-multiple-lesion group (MON group: with more than one ON lesion).</p> <p>Results</p> <p>Local intra-osseous perfusion index was found lower in either ON⁺or MON group when compared to either ON^-or SON group, whereas the marrow fat cells number and area were much larger in either ON⁺or MON group as compared with ON^-and SON group. The adipogenic differentiation ability of MSCs and PPARγ2 expression in either ON⁺or MON group were elevated significantly as compared with either ON^-or SON group.</p> <p>Conclusion</p> <p>These findings support our hypothesis that there is a close association between elevated adipogenesis and steroid-associated osteonecrosis development.</p

Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC)

<p>Abstract</p> <p>Background</p> <p>Protein used in medicine, e.g. interferon, are immunogenic and quickly broken down by the body. Pegylation is a recognized way of preserving their integrity and reducing immune reactions, and works well with enzymes used to degrade amino acids, a recent focus of attention in controlling cancer growth. Of the two arginine-degrading enzymes being explored clinically, arginine deiminase is a decidedly foreign mycoplasm-derived enzyme, whereas human arginase 1 is a native liver enzyme. Both have been pegylated, the former with adjuncts of 20 kD, the latter with 5 kD PEG. Pegylation is done by several different methods, not all of which are satisfactory or desirable.</p> <p>Methods</p> <p>The preparation of novel polyethylene glycol (PEG) derivatives for modifying proteins is described, but directed specifically at pegylation of recombinant human arginase 1 (rhArg1). rhArg1 expressed in <it>Escherichia coli </it>was purified and coupled in various ways with 5 different PEG molecules to compare their protective properties and the residual enzyme activity, using hepatocellular cell lines both in vitro and in vivo.</p> <p>Results</p> <p>Methoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA 5,000) coupled with very high affinity under mild conditions. The resulting pegylated enzyme (rhArg1-peg_{5,000 mw}) had up to 6 PEG chains of 5K length which not only protected it from degradation and any residual immunogenicity, but most importantly let it retain >90% of its native catalytic activity. It remained efficacious in depleting arginine in rats after a single ip injection of 1,500 U of the conjugate as the native enzyme, plasma arginine falling to >0.05 μM from ~170 μM within 20 min and lasting 6 days. The conjugate had almost the same efficacy as unpegylated rhArg1 on 2 cultured human liver cancer (HCC) cell lines. It was considerably more effective than 4 other pegylated conjugates prepared.</p> <p>Conclusion</p> <p>Valuable data on the optimization of the pegylation procedure and choice of ligand that best stabilizes the enzyme arginase 1 are presented, a protocol that should equally fit many other enzymes and proteins. It is a long lasting arginine-depleting enzyme in vivo which will greatly improve its use in anti-cancer therapy.</p

Constitutional Flavonoids Derived from Epimedium Dose-Dependently Reduce Incidence of Steroid-Associated Osteonecrosis Not via Direct Action by Themselves on Potential Cellular Targets

Intravascular-thrombosis and extravascular-lipid-deposit are the two key pathogenic events considered to interrupt intraosseous blood supply during development of steroid-associated osteonecrosis (ON). However, there are no clinically employed agents capable of simultaneously targeting these two key pathogenic events. The present experimental study demonstrated that constitutional flavonoid glycosides derived from herb Epimedium (EF, composed of seven flavonoid compounds with common stem nuclear) exerted dose-dependent effect on inhibition of both thrombosis and lipid-deposition and accordingly reducing incidence of steroid-associated ON in rabbits, which was not via direct action by themselves rather by their common metabolite on potential cellular targets involved in the two pathogenic pathways. The underlying mechanism could be explained by counteracting endothelium injury and excessive adipogenesis. These findings encourage designing clinical trials to investigate potential of EF in prevention of steroid-associated ON

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Proteomic study of Burkholderia sp. MBA4 in the degradation of haloacids

published_or_final_versionabstractBiological SciencesDoctoralDoctor of Philosoph

Supernate soluble TM in endotoxin induction group (A) and optical density in destained oil red O staining in steroid induction group (B) was significantly higher than the corresponding control group, respectively, whereas Icaritin dose-dependently lowered supernate soluble TM (A) and optical density in destained oil red O staining (B) when compared to the corresponding induction group, respectively.

<p>However, no differences were found between the seven parent flavonoids in EF at the concentration of 10⁻¹⁴ M and corresponding induction group both in supernate soluble TM (C) and optical density in destained oil red O staining (D). Note: * P<0.05 for comparison with the induction group. # P<0.05 for comparison with the low dose group.</p

Hematology/cytology/MRI data analysis.

<p>There was no significant change from baseline in ALT (A) and AST (B) when compared to the CON group. Significantly increased TM (C) from baseline in the CON group was attenuated in the L-EF group or prevented in both the M-EF and H-EF group at week 1 post induction, and adipocyte positive colonies (D) in the CON group were attenuated in the L-EF group or prevented in both the M-EF and H-EF group after induction. In addition, the significantly decreased PEP (E) from baseline in the CON group was attenuated in the L-EF group or prevented in both the M-EF and H-EF group at week 1 post induction. Note: * P<0.05 for comparison with CON; # P<0.05 for comparison with baseline. • CON group; ⧫ L-EF group; ▪ M-EF group; ▴ H-EF group.</p

Seven major flavonoid compounds are identified in <i>Epimedium</i>-derived Flavonoids (EF).

<p>The common structure is 8-prenylkaempferol. R1 and R2 are substituted by glucose, rhamnose, or xylose, and R3 is replaced by methyl.</p