Molecular Weight HA Research Articles

In Situ Cross-Linkable High Molecular Weight Hyaluronan−Bisphosphonate Conjugate for Localized Delivery and Cell-Specific Targeting: A Hydrogel Linked Prodrug Approach

We present here a novel synthesis route to functionalize high molecular weight hyaluronan (HMW-HA) with a hydrazide group and a bioactive ligand, namely bisphosphonate (BP). For this purpose, a new symmetrical self-immolative biscarbazate linker has been devised. The hydrazide group was used to form hydrazone cross-linked hydrogel upon treating with previously described aldehyde modified hyaluronan. The 1:1 weight ratio of these two polymers gave hydrogel in less than 30 s. In this communication we present the first in vitro results showing that even though HA can target CD44 positive cancer cells (HCT-116), receptor mediated endocytosis could only occur by cleavage of high molecular weight HA with an ubiquitous enzyme, hyaluronidase (Hase). The cancer cells are known to overexpress CD44 receptors and also increase the hyaluronidase activity in vivo. Thus the pro-drug design, based on drug conjugation to HMW-HA, represents a new drug delivery platform where the drug potency is triggered by Hase mediated degradation of the HA-drug conjugate. We have successfully demonstrated that the cross-linkable HA-BP conjugate first undergoes Hase-mediated scission to the fragments of suitable sizes so as to be internalized by CD44 positive cells. The specificity of this targeting was proven by comparing the results with less CD44 positive HEK-293T cells. The localized delivery of such drugs at the surgical resection site opens up avenues to control tumor recurrence after removal of the tumor. In the form of hydrogel it would prevent systemic exposure of the drug and would allow its controlled release.

Investigation of the swelling behavior of crosslinked hyaluronic acid films and hydrogels produced using homogeneous reactions

AbstractHyaluronic acid (HA) has been crosslinked in solution with glutaraldehyde (GTA), 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC), poly(ethylene glycol) diglycidylether (EX 810), and divinyl sulfone (DVS) to form hydrogels. Flory‐Rehner calculations were used to determine molecular weight between crosslinks (Mc), the crosslink density (Ve), and mesh size (ε) of crosslinked hydrogels after 24‐h swelling in distilled water. Generally, lower molecular weight films gave rise to decreased molecular weights between crosslinks as well as increased effective crosslink densities and decreased mesh size. The effects of pH and salt concentration were evaluated. Use of lower molecular weight HA gave rise to decreased molecular weights between crosslinks as well as increased effective crosslink densities and decreased mesh size. Water diffusion coefficients were measured for DVS and GTA hydrogels and were found to be 1.4 × 10−10 and 1.8 × 10−10 m2 s−1. Autocrosslinked and HA polyethyleneimine gels were also produced but had very limited stabilities compared with the covalently crosslinked materials. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

The role of low molecular weight hyaluronan in the pathogenesis of monocrotaline‐induced pulmonary hypertension

Extracellular matrix degradation products, such as low molecular weight hyaluronan (LMW HA), have been shown to stimulate the innate inflammatory response and could play a fundamental role in the pathogenesis of pulmonary hypertension (PH). The role of LMW HA in experimental, monocrotaline (MCT)‐induced PH was investigated in Fisher 344 rats. Twenty‐four days after MCT injection (0.7mg/kg), right ventricular systolic pressure was markedly elevated in MCT treated animals (62.9 ± 5.6 mmHg) when compared to no MCT controls (24.3 ± 0.8 mmHg). Quantification of total lung HA by ELISA revealed increased HA in MCT treated animals when compared to controls (248+/−71 versus 154+/−17 ng HA/mg dry lung). This data was supported by histological analysis of lung sections, which show elevated levels of HA in association with regions of vascular occlusion as well as with inflammatory cell infiltrates. Molecular weight analysis of lung‐derived HA by agarose gel electropheresis illustrated that MCT‐induced PH is associated with an accumulation of LMW HA (>5×105 Da) and a corresponding decrease in high molecular weight HA (∼4×106 Da). Gene expression analysis of the hyaluronan synthase (HAS) genes revealed that MCT‐induced PH corresponds with increased expression of HAS1 and HAS2 (1.67+/−0.14 and 3.37+/−0.55 fold increases vs. normal, respectively) and a decrease in HAS3 expression (4.86+/−0.16 fold decrease vs. normal).

Development, molecular composition and freeze tolerance of bovine embryos cultured in TCM-199 supplemented with hyaluronan

Hyaluronan (HA) is glycosaminoglycan that is present from the start of embryonic development and its role and concentration increases with embryo development. The objective of this study was to evaluate if the presence of HA in TCM-199 culture medium had an effect on the development and quality of bovine embryos. There was no effect of HA on the total number of zygotes developing to blastocysts on day 7, however more expanded and hatched blastocyst stages were observed on days 8 and 9 in the group supplemented with HA (p<0.05). Following freeze/thawing, significantly more (p<0.05) embryos cultured in medium supplemented with HA hatched than those cultured in TCM-199 alone or those with BSA. Medium supplemented with HA and BSA significantly increased the level of expression of glucose metabolism Glut-1 gene and embryo compaction Cx43 gene (p<0.05), and had no effect on Glut-5 and IGF-II expression. In addition, HA presence in culture decreased the level of expression of apoptosis Bax and oxidative stress SOX genes (p<0.05). There was significant difference in total number of nuclei between TCM-199 medium only and the remaining media containing BSA or HA plus BSA, between which there was no difference. In summary, our results indicate that the addition of high molecular weight HA to TCM-199 medium that contains BSA on day 4 of culture improved embryo development to hatching and hatched blastocysts and the quality of produced embryos, which were superior to embryos cultured without HA addition.

Expression and activity of Runx2 mediated by hyaluronan during chondrocyte differentiation

During endochondral ossification, the production of hyaluronan (HA) is strictly and selectively regulated by chondrocytes, with a temporal peak at the hypertrophic stage. This study was conducted to clarify the effects of HA on expression and activity of runt-related gene 2 (Runx2), a potent transcription factor for chondrocyte differentiation in hypertrophic chondrocytes. Immature chondrocytes from an ATDC5 cell line were cultured and differentiated in DMEM/Ham's F12 with pre-defined supplements. Using real-time PCR, the gene expressions of type II collagen, MMP-13, HAS2, and Runx2 in cultured chondrocytes were analysed from days 0 to 18 of cell differentiation. The activity and expression of Runx2 in hypertrophic chondrocytes were analysed after the treatment with HA oligosaccharide (HAoligo) using AML-3/Runx2 binding, real-time PCR and Western blot analysis. The effects of pre-incubation of anti-CD44 antibody on Runx2 expression were also examined. Expression of type X collagen and Runx2 mRNAs reached a maximum at the terminal differentiation of chondrocytes. The activity and expression of Runx2 was significantly inhibited in hypertrophic chondrocytes treated with HAoligo compared to the untreated controls. High molecular weight-HA did not affect the expression or activity of Runx2. The expression of Runx2 mRNA was significantly decreased in hypertrophic chondrocytes treated with anti-CD44 antibody. These results suggest that HAoligo may affect the terminal differentiation of chondrocytes during the endochondral ossification by inhibiting the expression and activity of Runx2.

The antioxidant effect exerted by TGF-1β-stimulated hyaluronan production reduced NF-kB activation and apoptosis in human fibroblasts exposed to FeSo4 plus ascorbate

Previous studies suggest that Transforming growth factor-1beta (TGF-1beta) administration in human fibroblasts exposed to oxidative stress is able to modulate hyaluronan synthases (HASs). HAS modulation in turn increases high molecular weight (Hyaluronan) HA concentration. Nuclear factor kB (NF-kB) is a response transcription factor involved in inflammation and acts by enabling the expression of certain detrimental molecules. Caspases are specific proteases responsible for regulating and programming cell death. HA at medium molecular weight together with chondroitin-4-sulphate proved to be effective on NF-kB and caspases. We investigated whether the protective effect afforded by the high molecular weight HA produced by TGF-1beta treatment has any effect on NF-kB and apoptosis activation in fibroblast cultures exposed to oxidative stress. Generation of free radicals gives rise to cell death, increases lipid peroxidation, activates NF-kB, reduces its cytoplasmic inhibitor IkBalpha, augments caspase-3 and caspase-7 gene expression and their relative protein activity, and depletes catalase (CAT) and glutathione peroxidase (GPx). Treatment of fibroblasts with TGF-1beta 12 h before inducing oxidative stress greatly increased HA levels, ameliorated cell survival, inhibited lipid peroxidation, blunted NF-kB translocation, normalized IkBalpha protein, reduced caspase gene expression and protein levels, and restored the endogenous antioxidants CAT and GPx. Since it was previously reported that antioxidants can work as inhibitors of NF-kB and apoptosis induction we can hypothesize that endogenous HA, by inhibiting lipid peroxidation, may block a step whereby free radical activity converges in the signal transduction pathway leading to NF-kB and caspase activation.

SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum

CD44-hyaluronan (HA) interaction is involved in diverse physiological and pathological processes. Regulation of interacting avidity is well studied on CD44 but rarely on HA. We discovered a unique covalent modification of HA with a protein, SHAP, that corresponds to the heavy chains of inter-alpha-trypsin inhibitor family molecules circulating in blood. Formation of the SHAP.HA complex is often associated with inflammation, a well known process involving the CD44-HA interaction. We therefore examined the effect of SHAP on the CD44-HA interaction-mediated lymphocyte adhesion. Under both static and flowing conditions, Hut78 cells (CD44-positive) and CD44-transfected Jurkat cells (originally CD44-negative) adhered preferentially to the immobilized SHAP.HA complex than to HA. The enhanced adhesion is exclusively mediated by the CD44-HA interaction, because it was inhibited by HA, but not IalphaI, and was completely abolished by pretreating the cells with anti-CD44 antibodies. SHAP appears to potentiate the interaction by increasing the avidity of HA to CD44 and altering their distribution on cell surfaces. Large amounts of the SHAP.HA complex accumulate in the hyperplastic synovium of rheumatoid arthritis patients. Leukocytes infiltrated to the synovium were strongly positive for HA, SHAP, and CD44 on their surfaces, suggesting a role for the adhesion-enhancing effect of SHAP in pathogenesis.

Overexpression of Hyaluronan Synthase 2 Alters Hyaluronan Distribution and Function in Proximal Tubular Epithelial Cells

The functional consequences of increased renal cortical hyaluronan that is associated with both acute injury and progressive scarring are unclear. The aim of this study was to characterize hyaluronan synthase-2 (HAS2)-driven HA synthesis and determine its effect on renal proximal tubular epithelial cell (PTC) function, because this is known to be the inducible form of HA synthase in this cell type. Overexpression of HAS2 mRNA increased HA generation, which in the supernatant predominantly was HA of large molecular weight, whereas there was an increase in low molecular weight HA in cell-associated fractions. This was associated with increased expression of hyaluronidases, inhibition of HA cable formation concurrent with reduction in HA-dependent monocyte binding, and increased pericellular HA matrix. Overexpression of HAS2 led to enhanced cell migration. HA can be modified by the covalent attachment of heavy chains that are derived from the serum protein inter-alpha-inhibitor (IalphaI), a process that is known to be catalyzed by TNF-alpha-stimulated gene 6 (TSG-6; an inflammation-associated protein). Enhanced migration was abrogated by blocking antibodies to either IalphaI or TSG-6. Addition of recombinant full-length TSG-6 (TSG-6Q) or TSG-6Q_Y94F, a mutant variant with impaired HA binding, increased cell migration. Both of these proteins were able to mediate the covalent transfer of heavy chains, from IalphaI and pre-alpha-inhibitor, onto HA. Addition of the isolated TSG-6-Link module (Link_TSG-6), which binds HA but is unable to form covalent complexes with IalphaI/pre-alpha-inhibitor, had no effect on migration, suggesting that TSG-6-mediated formation of heavy chain-HA complexes is critical in the formation of a pericellular HA matrix.

Ultrafiltration of water containing natural organic matter: heavy metal removing in the hybrid complexation–ultrafiltration process

The efficiency of heavy metal removal from water containing humic substances (HS) has been investigated by means of ultrafiltration. The rejection coefficient of Pb (II), Cu (II), Ni (II) and Co (II) in the presence of humic (HA) and fulvic acids (FA) was studied for different composition of feed solution (pH, concentration of metal ions and background electrolytes) and cellulose acetate ultrafiltration membranes of different molecular weight cut-off. HS rejection increases with pH due to conformational transformation of ionizable molecules. The interactions of HS with multi-charged metals do not practically affect the HS retention. Involvement of heavy metals ions into complexation with HS provides effective removal of trace metals from solutions as a result of complex retention. The metal rejection increases with pH growth and in neutral/alkaline solutions it exceeds 80%. Thus, ultrafiltration is an effective method for simultaneous removal of HS and heavy metals harnessing the principle of reagent-reinforced ultrafiltration. In conformity with this principle degree of heavy metal rejection improves with an increase in HS–Me ratio due to increase in completeness of metal binding. HS–Me ratio also is responsible for phase-dispersion state of the solution initiating a transition of intra-molecular complexes to inter-molecular ones at higher concentrations of metal ions with respect to higher molecular weight HA. This leads to an aggregation of HA–Me complexes and decrease in the trans-membrane flux. FA do not tend to form inter-molecular aggregates.

The effect of hyaluronan on osteoblast proliferation and differentiation in rat calvarial-derived cell cultures.

Hyaluronan (or hyaluronic acid, HA) is an essential component of extracellular matrices. It interacts with other macromolecules and plays a predominant role in tissue morphogenesis, cell migration, differentiation, and adhesion. The cell signaling functions of HA are mediated through the CD-44 receptor and are dependent upon the molecular weight of the polymer. We hypothesized that an HA of appropriate molecular weight alone in optimal concentration may induce osteoblast differentiation and bone formation. Enzyme-digested calvarial-derived mesenchymal cells from 2-day-old newborn rats were cultured with the addition of HA of three different molecular weights (2300, 900, and 60 kDa). We added, 0.5, 1.0, and 2.0 mg/mL HA for each molecular weight to the medium at the first plating of cells. After 7 to 20 days in culture, cell proliferation and differentiation were evaluated by measuring thymidine incorporation, alkaline phosphatase activity, and osteocalcin gene expression. The effects of HA on bone formation were examined by using Alizarin red staining for mineralization. The results showed that low molecular weight HA (60 kDa) significantly stimulated cell growth, increased osteocalcin mRNA expression in a dose-dependent manner, but showed no apparent effects on alkaline phosphatase activity and bone mineralization. On the other hand, high-weight HA (900 and 2,300 kDa) significantly increased all the parameters examined, particularly alkaline phosphatase activity, in a dose-dependent manner and stimulated cell mineralization to 126% and 119% of the controls, respectively, in the 1.0 mg/mL dose. Our findings suggest that HA has a molecular weight-specific and dose-specific mode of action that may enhance the osteogenic and osteoinductive properties of bone graft materials and substitutes due to its stimulatory effects on osteoblasts.

The usefullness of hyaluronate injection in patients with RA

In this seminar, we reported the usefulness of hyaluronate injection in clinical application with patients of RA. In basic research areas, however, the mechanisms responsible for the reported beneficial clinical effects of these treatments remain unclear. There is accumulating experimental evidence to show that the receptors of HA, eg, CD-44, accelerate the inflammation of joint. Administration high molecular weight HA may modulate CD-44 sensitive macromolecular in joint inflammation in part to reduce the activity of proteinases such as MMP-9.

Modulation of Fibrinolytic System Components in Mesothelial Cells by Hyaluronan

Hyaluronan (HA) is an important extracellular matrix component and is involved in fluid homeostasis, tissue repair, and response to infections. Previous studies have shown that supplementation of dialysis fluid with high molecular weight HA may have a positive impact on peritoneal solute and fluid transport characteristics. In the present study, we investigated the impact of HA on the synthesis of tissue-type plasminogen activator (t-PA) and its inhibitor, plasminogen activator inhibitor type 1 (PAI-1) in cultured human peritoneal mesothelial cells (MC). Cultured human peritoneal MC isolated from omental tissue were used for the experiments. Concentrations of t-PA and PAI-1 antigens were measured in conditioned media of confluent MC using ELISA. Northern blot analysis was performed to investigate mRNA expression of t-PA, PAI-1, and low-density lipoprotein receptor-related protein. Hyaluronan in a concentration as suggested for supplementation of dialysis fluid (10 mg/dL) did not have a significant impact on the synthesis of t-PA or PAI-1 in human MC. However, incubation of MC with higher concentrations of HA (30-1000 mg/dL) resulted in a concentration- and time- (8- 48 hours) dependent decrease in t-PA antigen release and mRNA expression. In contrast, PAI-1 antigen secretion was distinctly but not significantly increased in the presence of HA. The expression of t-PA and PAI-1 in MC was not affected by low concentrations of HA. Therefore, it is reasonable to assume that supplementation of dialysis fluid with HA (10 mg/dL) will not decrease mesothelial fibrinolytic activity. Only high concentrations (> 50 mg/dL) may disturb the balance between intraperitoneal generation and degradation of fibrin by decreasing t-PA synthesis.

Study of Hyaluronan-Binding Proteins and Receptors Using Iodinated Hyaluronan Derivatives

This chapter detailed methodology for the purification of high molecular weight HA, as well as procedures to fragment the HA to prepare large oligosaccharides in the range of 40,000-80,000 Da. The aforementioned procedures used to prepare HA-alkylamine and HA-Bolton-Hunter adducts, as well as 125I-labeled HA, have been very reproducible, and the latter preparations are of adequate length to retain high-affinity interactions and specific binding, e.g., to human fibrinogen and HARE. For example, we were able to isolate, characterize, and clone the rat HARE using 125I-labeled HA initially with the dot blot assay to monitor solubilization and partial purification, and later with the ligand blot assay, to identify the protein after SDS-PAGE. The ligand blot assay enabled us to determine that HARE is actually present as two discrete isoreceptors of different molecular masses. These techniques should provide a means to analyze purification strategies and to characterize additional HA receptors and binding proteins involved in a variety of physiologic processes.

Hyaluronan is not just a goo!

Hyaluronan is not just a goo!