Heavy chain transfer by tumor necrosis factor-stimulated gene 6 to the bikunin proteoglycan.

We present data that hyaluronan (HA) polysaccharides, about 14-86 monosaccharides in length, are capable of accepting only a single heavy chain (HC) from inter-α-inhibitor via transfer by tumor necrosis factor-stimulated gene 6 (TSG-6) and that this transfer is irreversible. We propose that either the sulfate groups (or the sulfation pattern) at the reducing end of the chondroitin sulfate (CS) chain of bikunin, or the core protein itself, enables the bikunin proteoglycan (PG) to accept more than a single HC and permits TSG-6 to transfer these HCs from its relatively small CS chain to HA. To test these hypotheses, we investigated HC transfer to the intact CS chain of the bikunin PG, and to the free chain of bikunin. We observed that both the free CS chain and the intact bikunin PG were only able to accept a single HC from inter-α-inhibitor via transfer by TSG-6 and that HCs could be swapped from the bikunin PG and its free CS chain to HA. Furthermore, a significant portion of the bikunin PG was unable to accept a single heavy chain. We discuss explanations for these observations, including the intracellular assembly of inter-α-inhibitor. In summary, these data demonstrate that the sulfation of the CS chain of bikunin and/or its core protein promote HC transfer by TSG-6 to its relatively short CS chain, although they are insufficient to enable the CS chain of bikunin to accept more than one HC in the absence of other cofactors.

Irreversible heavy chain transfer to chondroitin.

We have recently demonstrated that the transfer of heavy chains (HCs) from inter-α-inhibitor, via the enzyme TSG-6 (tumor necrosis factor-stimulated gene 6), to hyaluronan (HA) oligosaccharides is an irreversible event in which subsequent swapping of HCs between HA molecules does not occur. We now describe our results of HC transfer experiments to chondroitin sulfate A, chemically desulfated chondroitin, chemoenzymatically synthesized chondroitin, unsulfated heparosan, heparan sulfate, and alginate. Of these potential HC acceptors, only chemically desulfated chondroitin and chemoenzymatically synthesized chondroitin were HC acceptors. The kinetics of HC transfer to chondroitin was similar to HA. At earlier time points, HCs were more widely distributed among the different sizes of chondroitin chains. As time progressed, the HCs migrated to lower molecular weight chains of chondroitin. Our interpretation is that TSG-6 swaps the HCs from the larger, reversible sites on chondroitin chains, which function as HC acceptors, onto smaller chondroitin chains, which function as irreversible HC acceptors. HCs transferred to smaller chondroitin chains were unable to be swapped off the smaller chondroitin chains and transferred to HA. HCs transferred to high molecular weight HA were unable to be swapped onto chondroitin. We also present data that although chondroitin was a HC acceptor, HA was the preferred acceptor when chondroitin and HA were in the same reaction mixture.

Irreversible heavy chain transfer to hyaluronan oligosaccharides by tumor necrosis factor-stimulated gene-6.

The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).

Three dimensional culture of fresh and vitrified mouse pre-antral follicles in a hyaluronan-based hydrogel: a preliminary investigation of a novel biomaterial for in vitro follicle maturation.

BACKGROUND: Folliculogenesis within the ovary requires interaction between somatic cell components and the oocyte. Maintenance of 3-dimensional (3-D) architecture and granulosa-oocyte interaction may be critical for successful in vitro maturation of follicles. Testing of novel biomaterials for the 3-D culture of follicles may ultimately lead to a culture model that can support the longer in vitro culture intervals needed for in vitro maturation of human oocytes from ovarian tissue biopsies. METHODS: A novel tyramine-based hyaluronan (HA) hydrogel was tested for its biocompatibility with ovarian follicles. The HA was prepared at concentrations from 2 to 5 mg/ml. HA hydrogel was also formulated and tested with matrix proteins (ECM). Enzymatically isolated pre-antral follicles from the ovaries of 10-12 day SJL pups were divided amongst control (CT) and HA treatments. The growth of both fresh and vitrified follicles was assessed after encapsulation in the hydrogel. The basal culture medium was MEM alpha supplemented with FSH, LH, ITS and 5% FBS. Maturation was triggered by addition of hCG and EGF after in vitro culture (IVC). Outcome parameters monitored were follicle morphology, survival after IVC, antrum formation, GVBD and MII formation. Differences between treatments were analyzed. RESULTS: HA and ECM-HA encapsulated follicles looked healthy and maintained their 3-D architecture during IVC. In control cultures, the follicles flattened and granulosa:oocyte connections appeared fragile. Estradiol secretion per follicle was significantly higher by Day 12 in ECM-HA compared to HA or CT (4119, 703 and 1080 pg/ml, respectively). HA and ECM-HA cultured follicles had similar survival rates (62% and 54%, respectively), percent GV breakdown (96-97%), MII formation (47-48%) and oocyte diameters at the end of IVC. Control cultures differed significantly in percent GVBD (85%) and MII formation (67%) . Vitrified-warmed follicles encapsulated in HA had an oocyte maturation rate to MII of 54% as compared to 57% in non-embedded follicles. CONCLUSIONS: Initial testing of this new and unique HA-based hydrogel was quite promising. The ease of follicle encapsulation in HA, its optical transparency and ability to be molded combined with its support of follicle growth, estradiol secretion and resumption of meiosis make this HA-hydrogel particularly attractive as model for 3-D ovarian follicle culture.

Identification of unique synergistic drug combinations associated with downexpression of survivin in a preclinical breast cancer model system.

An in-vitro 72-h assay using median effect analysis and curve shift analysis was used to evaluate the utility of potentially clinically useful combinations of agents for synergism or antagonism. Six human breast cancer cell lines, both receptor rich and receptor poor, were studied.Panobinostat (LBH-589), a pan histone deacetylase inhibitor with a multitude of biological effects, exhibits time-dependent synergistic effects in breast cancer cell lines with docetaxel, doxorubicin, or gemcitabine in clinically relevant concentrations. Survivin expression was markedly downregulated in the presence of panobinostat with gemcitabine. Bortezomib, a proteasome inhibitor,markedly enhanced the cytotoxic effects of panobinostat combined with gemcitabine. Panobinostat did not demonstrate universal enhancement of cytotoxic drugs,and therefore, synergy was dependent on the second agent selected. No synergy was noted with anti-Her2 agents in Her2 overexpressing cell lines. Metformin combined with panobinostat demonstrated no synergy in this test system. These effects were confirmed by an apoptosis assay and caspase-3 production. A positive drug interaction was identified. The triplet of panobinostat with either doxorubicin/carboplatin or gemcitabine/carboplatin was especially potent in all cell lines. As all these agents are clinically available, further studies of the potent combinations are warranted.

A synthetic cartilage extracellular matrix model: hyaluronan and collagen hydrogel relaxivity, impact of macromolecular concentration on dGEMRIC.

OBJECTIVE: To develop and characterize the MR properties of a synthetic model for cartilage extra-cellular matrix using hydrogels and to determine the concentration dependence of spin-lattice (T1) and spin-spin (T2) relaxation times of hydrogels and their glycosaminoglycan and collagen components in the presence and absence of gadopentetate dimeglumine (Gd-DTPA) for use in dGEMRIC. MATERIALS AND METHODS: T1 and T2 measurements were made at 3 Tesla on a range of gelatin (i.e., collagen) and hyaluronan (i.e., glycosaminoglycan) solutions (6.25-100 g/l), alone, together in a composite, and as dityramine-bridged hydrogels. Relaxivity was calculated as a function of macromolecular concentration. RESULTS: Even at the highest concentrations, gelatin and hyaluronan solutions had T1 and T2 values significantly larger than those reported for cartilage. Only composite hydrogels with gelatin and hyaluronan concentrations naturally found in cartilage resulted in T1 values, but not T2 values, representative of cartilage. Relaxivities were slightly dependent on both hyaluronan concentration (R1 = 0.0027 l g(-1) s(-1); R2 = 0.025 l g(-1) s(-1)) and gelatin concentration (R1 = 0.0032 l g(-1) s(-1); R2 = 0.020 l g(-1) s(-1)) alone and as a composite (R1 = 0.0068 l g(-1) s(-1); R2 = 0.101 l g(-1) s(-1)). Gd-DTPA relaxivities were dependent upon macromolecular concentration and varied by 14-32% (R1 = 4.24 to 5.55 mM(-1) s(-1); R2 = 4.60 to 6.27 mM(-1) s(-1)) over the range of cartilage biochemistry. CONCLUSIONS: Without the contrast agent, hyaluronan and gelatin, alone or in a composite, have a very small impact on the relaxivities of the model system. The impact on R1 was approximately tenfold less than on R2. In contrast, macromolecular concentrations above 50 g/l significantly impacted Gd-DTPA relaxivity and should be accounted for when measuring the glycosaminoglycan content of cartilage in vivo using dGEMRIC.

The effect of hyaluronan hydrogel on fat graft survival.

BACKGROUND: Autologous fat transplantation is a common technique for soft tissue augmentation in aesthetic and reconstructive surgery; however, the degree of fat graft take can be unpredictable. Hyaluronan has been shown to be a promising cell carrier in adipose tissue engineering. OBJECTIVES: The authors investigate the effect of a hyaluronan hydrogel on fat graft survival, angiogenesis, and volume maintenance in a rat model. METHODS: Fat was harvested from the groins of 27 rats, processed, and injected beneath the animals' dorsums to form 2 grafts: 1 containing fat alone and 1 containing fat and hyaluronan hydrogel in a 1:1 mix (fat-HA). The grafts were scanned in vivo under high-resolution computed tomography at baseline and prior to euthanasia at 4, 12, and 20 weeks to measure total fat-HA graft volume as well as the volume of the fat component alone. Histological studies were performed after sacrifice to evaluate fat necrosis and blood vessel density. RESULTS: All grafts were clinically viable. Overall, fat necrosis was significantly reduced in the fat-HA grafts compared with the grafts containing fat alone (P < .001). This difference was most profound at 4 weeks (P = .008) but did not reach statistical significance at 12 and 20 weeks. At 12 weeks, blood vessel density in the fat-HA grafts was significantly greater than in the grafts containing fat alone (P = .016), but this did not reach statistical significance at 4 or 20 weeks. At 20 weeks, the fat component of the fat-HA graft had significantly less volume loss than the fat-alone graft (P = .008). CONCLUSIONS: When mixed with fat, hyaluronan hydrogel can improve early fat graft survival and may enhance vascularity and prolong volume maintenance.

The pharmacokinetics of therapeutic arsenic trioxide in acute promyelocytic leukemia patients.

Arsenic trioxide (ATO) treats Acute Promyelocytic Leukemia (APL). ATO is converted from inorganic arsenic (iAs) to methylated (MAs) and dimethylated (DMAs) metabolites, which are excreted in the urine. Methylation of iAs is important in detoxification, as iAs exposure is deleterious to health. We examined ATO metabolism in 25 APL patients, measuring iAs, MAs, and DMAs. Plasma total iAs increased after ATO administration, followed by a rapid decline, reaching trough levels by 4-6 h. We identified two patterns of iAs metabolism between 6 and 24 h after infusion: in Group 1, iAs increased and were slowly converted to MAs and DMAs, whereas in Group 2, iAs was rapidly metabolized. These patterns were associated with smoking and different treatments: ATO with all-trans retinoic acid (ATRA) alone vs. ATO preceded by ATRA and chemotherapy. Our data suggest that smoking and prior chemotherapy exposure may be associated with ATO metabolism stimulation, thus lowering the effective blood ATO dose.

Age-related differences in human skin proteoglycans.

Previous work has shown that versican, decorin and a catabolic fragment of decorin, termed decorunt, are the most abundant proteoglycans in human skin. Further analysis of versican indicates that four major core protein species are present in human skin at all ages examined from fetal to adult. Two of these are identified as the V0 and V1 isoforms, with the latter predominating. The other two species are catabolic fragments of V0 and V1, which have the amino acid sequence DPEAAE as their carboxyl terminus. Although the core proteins of human skin versican show no major age-related differences, the glycosaminoglycans (GAGs) of adult skin versican are smaller in size and show differences in their sulfation pattern relative to those in fetal skin versican. In contrast to human skin versican, human skin decorin shows minimal age-related differences in its sulfation pattern, although, like versican, the GAGs of adult skin decorin are smaller than those of fetal skin decorin. Analysis of the catabolic fragments of decorin from adult skin reveals the presence of other fragments in addition to decorunt, although the core proteins of these additional decorin catabolic fragments have not been identified. Thus, versican and decorin of human skin show age-related differences, versican primarily in the size and the sulfation pattern of its GAGs and decorin in the size of its GAGs. The catabolic fragments of versican are detected at all ages examined, but appear to be in lower abundance in adult skin compared with fetal skin. In contrast, the catabolic fragments of decorin are present in adult skin, but are virtually absent from fetal skin. Taken together, these data suggest that there are age-related differences in the catabolism of proteoglycans in human skin. These age-related differences in proteoglycan patterns and catabolism may play a role in the age-related changes in the physical properties and injury response of human skin.

The histone deacetylase inhibitor panobinostat demonstrates marked synergy with conventional chemotherapeutic agents in human ovarian cancer cell lines.

Although platinum based therapy has improved short term survival of patients with metastatic ovarian cancer, the majority of patients continue to relapse and eventually die of their disease. Currently, a plethora of agents are in development, but how to combine them to enhance efficacy remains largely empiric. We have used short in vitro culture of defined cell lines with application of promising agents and analysis for cell death using a MTT assay to identify potentially useful combinations. Using median effect analysis, curve shift analysis and apoptosis assays, we can identify when agents are synergistic or antagonistic when applied together. Up to three agents can be studied in combination. Using three cell lines: SK-OV3, CaOV-3, and ES-2 (a clear cell tumor), we have identified that panobinostat (LBH-589), a broad histone deacetylase inhibitor in clinical trials, demonstrates global synergy with gemcitabine, with paclitaxel, and additive to synergistic effects with 5'DFUR. The triplet of panobinostat, doxorubicin, and carboplatin is especially synergistic in these cell lines. These effects are cytotoxic and not cytostatic. As all these agents are used clinically, we have identified combinations which warrant further investigation.