In vivo multimodal imaging of hyaluronan-mediated inflammatory response in articular cartilage.

OBJECTIVE: One driving factor in the progression to posttraumatic osteoarthritis (PTOA) is the perpetuation of the inflammatory response to injury into chronic inflammation. Molecular imaging offers many opportunities to complement the sensitivity of current imaging modalities with molecular specificity. The goal of this study was to develop and characterize agents to image hyaluronan (HA)-mediated inflammatory signaling. DESIGN: We developed optical (Cy5.5-P15-1) and magnetic resonance contrast agents (Gd-DOTA-P15-1) based in a hyaluronan-binding peptide (P15-1) that has shown anti-inflammatory effects on human chondrocytes, and validated them in vitro and in vivo in two animal models of PTOA. RESULTS: In vitro studies with a near infrared (NIR) Cy5.5-P15-1 imaging agent showed a fast and stable localization of Cy5.5-P15-1 on chondrocytes, but not in synovial cells. In vivo NIR showed significantly higher retention of imaging agent in PTOA knees between 12 and 72 h (n = 8, Cohen's d > 2 after 24 h). NIR fluorescence accumulation correlated with histologic severity in cartilage and meniscus (ρ between 0.37 and 0.57, P < 0.001). By using in vivo magnetic resonance imaging with a Gd-DOTA-P15-1 contrast agent in 12 rats, we detected a significant decrease of T1 on injured knees in all cartilage plates at 48 h (-15%, 95%-confidence interval (CI) = [-18%,-11%]) while no change was observed in the controls (-2%, 95%-CI = [-5%,+1%]). CONCLUSIONS: This study provides the first in vivo evidence that hyaluronan-related inflammatory response in cartilage after injury is a common finding. Beyond P15-1, we have demonstrated that molecular imaging can provide a versatile technology to investigate and phenotype PTOA pathogenesis, as well as study therapeutic interventions.

The effect of molecular weight on hyaluronan's cartilage boundary lubricating ability--alone and in combination with proteoglycan 4.

OBJECTIVES: (1) assess the molecular weight dependence of hyaluronan's (HA) cartilage boundary lubricating ability, alone and in combination with proteoglycan 4 (PRG4), at physiological concentrations; (2) determine if HA and PRG4 interact in solution via electrophoretic mobility shift assay (EMSA). METHODS: The cartilage boundary lubricating ability of a broad range of MW HA (20 kDa, 132 kDa, 780 kDa, 1.5 MDa, and 5 MDa) at 3.33 mg/ml, both alone and in combination with PRG4 at 450 µg/ml, was assessed using a previously described cartilage-on-cartilage friction test. Static, µ(static, Neq), and kinetic, <µ(kinetic, Neq)>, were calculated. An EMSA was conducted with PRG4 and monodisperse 150 kDa and 1,000 kDa HA. RESULTS: Friction coefficients were reduced by HA, in a MW-dependent manner. Values of <µ(kinetic, Neq)> in 20 kDa HA, 0.098 (0.089, 0.108), were significantly greater compared to both 780 kDa, 0.080 (0.072, 0.088), and 5 MDa, 0.079 (0.070, 0.089). Linear regression showed a significant correlation between both µ(static, Neq) and <µ(kinetic, Neq)>, and log HA MW. Friction coefficients were also reduced by PRG4, and with subsequent addition of HA; however the synergistic effect was not dependent on HA MW. Values of <µ(kinetic, Neq)> in PRG4, 0.080 (0.047, 0.113), were significantly greater than values of PRG4+various MW HA (similar in value, averaging 0.040 (0.033, 0.047)). EMSA indicated that migration of 150 kDa and 1,000 kDa HA was retarded when combined with PRG4 at high PRG4:HA ratios. CONCLUSIONS: These results suggest alterations in HA MW could significantly affect synovial fluid's cartilage boundary lubricating ability, yet this diminishment in function could be circumvented by physiological levels of PRG4 forming a complex, potentially in solution, with HA.

Tapping mode atomic force microscopy of hyaluronan: extended and intramolecularly interacting chains.

The extracellular matrix polysaccharide hyaluronan has been examined by tapping mode atomic force microscopy. High molecular weight hyaluronan was deposited on mica from dilute aqueous solution and imaged in air. Long unbranched chains could be observed and were found to be compatible with the known covalent structure of hyaluronan. In addition, chains with evidence of intramolecular association were observed. In the simplest cases, the association took the form of loops stabilized by antiparallel double-stranded (probably double-helical) segments. In other cases, the polarity of the associated regions could not be determined. Extensive intramolecular association in long hyaluronan chains resulted in a fenestrated structure of the same type as that formed by intermolecular association at higher concentrations.

Nitric oxide degradation of heparin and heparan sulphate.

NO is a bioactive free radical produced by NO synthase in various tissues including vascular endothelium. One of the degradation products of NO is HNO2, an agent known to degrade heparin and heparan sulphate. This report documents degradation of heparin by cultured endothelial-cell-derived as well as exogenous NO. An exogenous narrow molecular-mass preparation of heparin was recovered from the medium of cultured endothelial cells using strong-anion exchange. In addition, another narrow molecular-mass preparation of heparin was gassed with exogenous NO under argon. Degradation was evaluated by gel-filtration chromatography. Since HNO2 degrades heparin under acidic conditions, the reaction with NO gas was studied under various pH conditions. The results show that the degradation of exogenous heparin by endothelial cells is inhibited by NO synthase inhibitors. Exogenous NO gas at concentrations as low as 400 p.p.m. degrades heparin and heparan sulphate. Exogenous NO degrades heparin at neutral as well as acidic pH. Endothelial-cell-derived NO, as well as exogenous NO gas, did not degrade hyaluronan, an unrelated glycosaminoglycan that resists HNO2 degradation. Peroxynitrite, a metabolic product of the reaction of NO with superoxide, is an agent that degrades hyaluronan; however, peroxynitrite did not degrade heparin. Thus endothelial-cell-derived NO is capable of degrading heparin and heparan sulphate via HNO2 rather than peroxynitrite. These observations may be relevant to various pathophysiological processes in which extracellular matrix is degraded, such as bone development, apoptosis, tissue damage from inflammatory responses and possible release of growth factors and cytokines.

Degradation of hyaluronan by peroxynitrite.

Treatment of high-molecular-weight hyaluronan (HA) with peroxynitrite at neutral pH (ONOO-/ONOOH) results in altered mobility on agarose gel electrophoresis, as well as reduced limiting viscosity number. Both effects are consistent with a reduction in HA molecular weight. HA is protected from peroxynitrite attack to varying extents by addition of alternate target molecules. Thiourea is extremely effective as a protective agent, dimethyl sulfoxide is moderately effective, while sodium benzoate and mannitol are slightly effective. A similar pattern of protection is observed when HA is degraded by hydroxyl radical generated by a metal ion/hydrogen peroxide system. On the basis of these observations, peroxynitrite is proposed to have hydroxyl radical-like activity in degrading HA.

An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution.

An electrophoretic method is described for determining the molecular weight distribution of hyaluronan (HA). The method involves separation of HA by electrophoresis on a 0.5% agarose gel, followed by detection of HA using the cationic dye Stains-All (3,3'-dimethyl-9-methyl-4,5,4'5'-dibenzothiacarbocyanine). The recommended sample load is 7 micrograms. Calibration of the method with HA standards of known molecular weight has established a linear relationship between electrophoretic mobility and the logarithm of the weight-average molecular weight over the range of approximately 0.2-6 x 10(6). The separated HA pattern may also be visualized after electrotransfer of HA from the agarose gel to a nylon membrane. The membrane may be stained with the dye alcian blue. Alternatively, specific detection of HA from impure samples can be achieved by probing the nylon membrane with biotin-labeled HA-binding protein and subsequent interaction with a streptavidin-linked gold reagent and silver staining for amplification. The electrophoretic method was used to analyze HA in two different liquid connective tissues. Normal human knee joint synovial fluid showed a narrow HA molecular weight distribution, with a peak at 6-7 x 10(6). Owl monkey vitreous HA also showed a narrow molecular weight distribution, with a peak at 5-6 x 10(6). These results agree well with available published data and indicate the applicability of the method to the analysis of impure HA samples which may be available in limited amounts.

Self-association of hyaluronate segments in aqueous NaCl solution.

The potential for self-association by hyaluronate (HA) chains in 0.15 M NaCl was investigated, using low molecular weight HA segments as a model system. HA segments were derived from the polymer by controlled enzymatic digestion, and purified by gel filtration chromatography. Seven samples of narrow molecular weight distribution were analyzed by sensitivity-enhanced polyacrylamide gel electrophoresis, and found to have the following weight-average numbers of repeating disaccharide units: A, 90; B, 51; C, 38; D, 31; E, 23; F, 18; G, 13. The segment preparations were studied in 0.15 M NaCl by capillary viscometry, low angle laser light scattering, and circular dichroism spectroscopy. The data indicate concentration-dependent intermolecular association of short segments, and a capability for intramolecular association (hairpin formation) by larger HA segments.

High-performance gel permeation chromatography of glycosaminoglycans. Column calibration by gel electrophoresis.

High-performance gel permeation chromatography and sensitivity-enhanced polyacrylamide gel electrophoresis (SE-PAGE) have been combined to investigate the chromatographic properties of structurally related glycosaminoglycans. Enzymatically digested samples of hyaluronate, chondroitin 4-sulfate and dermatan sulfate were fractionated on a column series of TSK G4000SW and TSK G2000SW, eluted with 0.15 M sodium chloride. Isolated fractions were subjected to sensitivity-enhanced polyacrylamide gel electrophoresis for determination of molecular weight. These procedures allowed the rapid development of column calibration profiles for each glycosaminoglycan, under a given set of chromatographic conditions. The profiles obtained in 0.15 M sodium chloride yielded the following data: (1) at equal degrees of polymerization, hyaluronic acid chains have an apparently smaller hydrodynamic radius than the sulfated polymers, and (2) at equal molecular weights, all three glycosaminoglycans have approximately equal hydrodynamic radii.

Combined alcian blue and silver staining of glycosaminoglycans in polyacrylamide gels: application to electrophoretic analysis of molecular weight distribution.

Oligomeric and polymeric fragments of glycosaminoglycans may be separated for rapid analysis by electrophoresis through a 10% polyacrylamide matrix. A ladder-like series of bands is observed, in which adjacent major bands correspond to species differing in chain length by one disaccharide unit. The component species are detected by a combined alcian blue and silver staining protocol. Detection limits are less than 50 ng per band, or approximately 2-5 micrograms total load for polydisperse samples. Densitometry of the stained gel may be used to determine molecular weight averages and distribution. The applicable molecular weight ranges are approximately 4000 to 100,000 for hyaluronate, or 1500 to 40,000 for chondroitin and dermatan sulfate samples of moderate charge density heterogeneity.

Cationic dye binding by hyaluronate fragments: dependence on hyaluronate chain length.

Sodium hyaluronate, digested with bovine testicular hyaluronidase, yielded a mixture of oligosaccharides with identical repeating disaccharide structures and differing molecular weights. The oligosaccharides were separated into a ladder-like series of bands by electrophoresis on a 10% polyacrylamide gel matrix. Coelectrophoresis of purified oligosaccharides has established that adjacent bands differ in chain length by one disaccharide unit. This procedure formed the basis for a rapid screening method in which the binding of cationic dyes by hyaluronate oligosaccharides may be assayed. As a function of chain length, the oligosaccharides showed a marked change in dye binding. Species containing less than seven repeating disaccharide units are not detected by any dye tested, even at very high sample loads. Larger oligosaccharides show an increase in dye binding. The chain length at which constant maximal dye binding is reached depends on the dye structure and solvent conditions, varying from approximately 12 to 30 disaccharide units. The hyaluronate fragments of sufficient chain length to duplicate polymer behavior should be useful models for the study of hyaluronate structure and interactions in solution.

Polyacrylamide-gel electrophoresis and Alcian Blue staining of sulphated glycosaminoglycan oligosaccharides.

Oligosaccharide fragments of glycosaminoglycans may be separated for rapid analysis by electrophoresis through a 10% polyacrylamide matrix. An extensive ladder-like set of bands is observed for partial testicular hyaluronidase digests of chondroitin 4- or 6-sulphate, and for dermatan sulphate. Co-electrophoresis of purified oligosaccharides has established that the major bands of these patterns represent fragments differing in chain length by one disaccharide unit, with the smallest fragments having the greatest mobility. Additional minor bands, representing heterogeneity in the repeating unit structure, are also observed. There are slight differences in the mobilities of oligosaccharides derived from the three major types of sulphated glycosaminoglycans. Alcian Blue is employed for visualization of the digest fragments. Sample loads of 5-10 micrograms per band appear optimum. The smallest oligosaccharide which may be stained by this method is the hexasaccharide. After consideration of this effect, a good correlation is found to exist between densitometric scans of the gel-electrophoretic patterns and gel-filtration chromatographic profiles based on uronic acid concentration.

1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: the amide proton environment.

The exchangeable amide protons of hyaluronic acid (HA) oligosaccharides and a higher-molecular-weight segment dissolved in H2O at pH 2.5 or 5.5 were examined by H NMR spectroscopy at 250 MHz. The HA segment preparation showed a single amide resonance, near the chemical shift for the amide proton of the monosaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-GlcNAc). Smaller HA oligosaccharides showed two or three separate amide proton resonances, corresponding in relative peak area to interior or end GlcNAc residues. The interior GlcNAc amide resonance occurred at the same chemical shift as the single resonance of the HA segment. For the end GlcNAc residues, linkage to D-glucuronopyranose (GlcUA) through C1 resulted in an upfield shift relative to the beta-anomer of GlcNAc, whereas linkage through C3 resulted in a downfield shift relative to the corresponding anomer of GlcNAc. These chemical-shift perturbations appeared to be approximately offsetting in the case of linkage at both positions. The amide proton vicinal coupling constant (ca. 9 Hz) was found to be essentially independent of chain length, residue position, or solution pH. These data favor a nearly perpendicular orientation for the acetamido group with respect to the sugar ring, little affected by linkage of GlcNAc to GlcUA. No evidence for the existence of a stable hydrogen bond linking the amide proton with the carboxyl(ate) oxygen of the adjacent uronic acid residue was found. The amide proton resonances for chondroitin, chondroitin 4-sulfate, and dermatan sulfate were compared to that of HA. The chemical shifts of these resonances deviated no more than 0.1 ppm from that of HA. A small dependence on the identity of the adjacent uronic acid residue was noted, based on the observation of two resonances for dermatan sulfate.

Dependence of mononucleosome deoxyribonucleic acid conformation on the deoxyribonucleic acid length and H1/H5 content. Circular dichroism and thermal denaturation studies.

Four mononucleosome preparations were isolated from micrococcal nuclease digests of chicken erythrocyte nuclei which differed in average deoxyribonucleic acid (DNA) length and in H1 and H5 content. The circular dichroism properties of the unperturbed mononucleosome preparations and the corresponding H1- and H5-depleted species demonstrate that the nucleoprotein spectra above 250 nm are all altered relative to protein-free DNA by the addition of a single negative band at 275 nm, similar to the band observed for psi-DNA. The quantitative analysis of the psi-type band intensity for any of the higher molecular weight unperturbed samples relative to core particle mononucleosomes yielded a constant number of DNA base pairs (approximately 140) contributing to this new band. Upon removal of H1 and H5 from the mononucleosome preparations which have sufficiently long linker DNA, the psi-type band intensity indicates an approximately 30 base pair reduction in the number of core DNA base pairs contributing to the altered circular dichroism properties. The psi-type band is proposed to be due to the compact DNA tertiary structure, i.e., the manner in which the DNA is wound around the histone core allowing interactions between adjacent turns of the superhelix. This interpretation implies that approximately 30 base pairs of core DNA are removed from the unique core tertiary structure when the linker DNA is not bound by H1 or H5. The circular dichroism analysis correlates well with the thermal denaturation properties of mononucleosomes. Removal of H1 and H5 causes an overall reduction in the thermal stability of both core and linker DNA. The degree of destabilization is greatest when the average DNA length is maximum. Some core DNA is lost from the highest temperature melting bands when histone-free DNA is present. These results indicate two regions of different conformational and thermodynamic stability in core DNA. The length of attached linker DNA and its histone content influence the two regions of the core to differing extents.

Circular dichroism analysis of mononucleosome DNA conformation.

Mononucleosomes were isolated from micrococcal nuclease digests of chicken erythrocyte nuclei. The circular dichroism properties of mononucleosome preparations, differing in average DNA length and in H1 and H5 content, demonstrate that the spectrum of chromatin is due only to the complete structure of its repeating subunits. The nucleoprotein spectra are all altered relative to protein-free DNA by the emergence of a single negative band at 275 nm, similar to the band observed for psi DNA. The intensity of the psi-type band depends on the proportion of DNA condensed in a specific manner. The psi-type band is proposed to be due to the compact DNA tertiary structure; i.e., the manner in which the DNA is wound around the histone core allowing interactions between adjacent turns of the superhelix. This interpretation attributes changes and variability in nucleoprotein circular dichroism spectra under different experimental conditions to alterations in DNA tertiary structure rather than secondary structure.