Removal Of Hyaluronan Research Articles

Binding of Trastuzumab to ErbB2 Is Inhibited by a High Pericellular Density of Hyaluronan

Although trastuzumab is an efficient drug, primary and acquired resistance is a challenging problem. The authors have previously shown in mouse xenograft experiments that masking ErbB2 by hyaluronan leads to diminished binding of the antibody and consequent resistance. In the current work, they correlated trastuzumab binding with the pericellular density of hyaluronan in ErbB2-overexpressing human breast cancer samples. A method for quantifying the relative binding of trastuzumab was developed involving constant and low-frequency background subtraction, segmenting the image to membrane and background pixels followed by evaluation of trastuzumab fluorescence, normalized with the expression level of ErbB2, only in the membrane. The normalized binding of trastuzumab showed a negative correlation with the pericellular density of hyaluronan (r = -0.52) with the effect being the most pronounced in the extreme cases (i.e., low and high hyaluronan densities predicted strong and weak binding of trastuzumab, respectively). Removal of hyaluronan by hyaluronidase digestion unmasked the trastuzumab binding epitope of ErbB2 demonstrated by a significantly increased normalized binding of the antibody. The results show that the accumulation of pericellular hyaluronan plays a crucial role in masking ErbB2.

Abstract 3796: Pegylated human recombinant hyaluronidase PH20 reduces solid tumor hypoxia

Abstract Tumor hypoxia, the pathophysiological result of the structural and functional disruption of the tumor microcirculation and the deterioration of normal diffusion geometry, is strongly associated with tumor proliferation and resistance to therapy, both chemotherapy and radiotherapy. Indeed, hypoxia-associated resistance to photon radiotherapy is a severe clinical problem, as the radiation doses required to achieve the same treatment effect in hypoxic tumors can be three times the doses required in normoxic tumors. The extracellular matrix (ECM) glycosaminoglycan hyaluronan (HA) accumulates to high levels in ∼30% of solid tumors. Since HA is highly hydrophilic, ECM HA accumulation is believed to contribute to the elevated interstitial fluid pressure (IFP) and subsequent tumor vessel constriction observed in solid tumors. This vascular constriction contributes to the pathologic hypoxia present in these tumors. PEGPH20, a pegylated human recombinant hyaluronidase PH20, when delivered intravenously in preclinical models has been shown to enzymatically remove tumor HA and decrease both tumor IFP and water, leading to increased tumor vascular perfusion and enhanced chemotherapeutic delivery (Thompson 2010). Here we aimed to extend these observations and determine whether PEGPH20 would both increase tumor blood flow and reduce tumor hypoxia following PEGPH20-mediated HA removal. Nude mice were inoculated with human BxPC-3 pancreatic cancer cells adjacent to the right tibial periosteum. When tumors reached 15-20 mm in diameter (nα8/group), mice were staged into two treatment groups: (1) vehicle control and (2) PEGPH20 monotherapy. Vehicle or PEGPH20 (4.5 mg/kg) was administered to animals intravenously twice weekly for one week. The hypoxyprobe pimonidazole (60 mg/kg, ip) was administered to animals two hours prior to sacrifice, and fluorescent carbocyanine (75 μL, iv) 5 minutes prior to sacrifice. At sacrifice, whole tumors were removed, bisected, embedded in OCT medium, and processed for immunohistochemistry (hypoxyprobe and CD31) or simply imaged microscopically (carbocyanine). Hypoxia, blood vessel position, and tumor perfusion were assessed using pimonidazole, CD31 and carbocyanine, respectively. Compound images were created and the spatial relationship between hypoxia and vasculature perfusion evaluated. Consistent with prior studies, tumor HA was depleted in the PEGPH20-treated tumors. Further, tumor vascular perfusion (carbocyanine) increased 86% (p=0.0007) and tumor hypoxia decreased by 66% (p=0.03) in PEGPH20-treated animals, relative to the vehicle controls. These findings suggest that PEGPH20-mediated reduction in tumor HA increases tumor perfusion, while concomitantly reducing tumor hypoxia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3796. doi:1538-7445.AM2012-3796

Abstract 2501: PEGylated human recombinant hyaluronidase (PEGPH20) enhances radiotherapy in hyaluronan-rich solid tumors

Abstract The accumulation of the glycosaminoglycan hyaluronan (HA) in the extracellular matrix (ECM) of solid tumors contributes to increased tumor interstitial fluid pressure IFP), localized edema and reduced intratumoral blood flow. We recently reported that sustained enzymatic depletion of tumor HA with a pegylated recombinant human hyaluronidase PH20 (PEGPH20) decreases tumor IFP and water content, while significantly increasing vascular perfusion within HA-rich prostate PC3 tumors (Thompson et al, 2010). As localized tissue hypoxia reduces the effectiveness of radiation therapy (Baumgartner et al, 1998), we hypothesized that PEGPH20 treatment would reduce tumor hypoxia, thereby radio-sensitizing tumor cells. To evaluate whether PEGPH20 sensitizes HA-rich tumors, radiotherapy resistant human prostate PC3 cells (2×106) were inoculated into the right tibia periosteum of nude mice (NCR nu/nu) and tumor growth monitored with a high resolution 3D ultrasound. When the tumors reached ∼800 mm3 (n ≥ 8/group), mice were staged into 4 treatment groups: (1) vehicle control; (2) mono-radiotherapy at 2.5 Gy, q3d; (3) PEGPH20 monotherapy, 4.5 mg/kg, i.v. q3d; or (4) PEGPH20 plus radiotherapy. To increase intra-tumoral blood flow, PEGPH20 was administrated 3 hours prior to radiation therapy. Histological evaluation of a subset of mice confirmed that HA was completely degraded 3 hours post- PEGPH20 treatment. At study Day 15, tumor growth inhibition (TGI) with radiation alone or PEGPH20 alone was 99.2% (p=0.009) and 71.4% (p=0.08), respectively, relative to vehicle treated animals. PEGPH20 plus radiotherapy inhibited tumor growth by 140.6% (p=0.0002), relative to vehicle treated animals. The mean tumor size of the PEGPH20 plus radiotherapy group was reduced to ∼550 mm3, with 3-out-of-8 partial regressors (i.e. tumor size decrease to 50% or less of tumor staging size). These findings suggest that PEGPH20-mediated HA removal, and subsequent increase in tumoral blood flow, may increase the efficacy of radiation therapy in hyaluronan-rich tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2501. doi:10.1158/1538-7445.AM2011-2501

Hyaluronan‐chondrocyte interactions mediate cell signaling pathways

Osteoarthritis is a degenerative process of articular cartilage. The focus of our project is to identify strategies to stimulate cartilage matrix repair. An intact, hyaluronan‐dependent pericellular matrix promotes the chondrocyte response to BMP‐7. Disruption of chondrocyte‐matrix interactions occurs during disease progression. The purpose of this study is to validate the function of CD44, the principal hyaluronan receptor, in mediating the effects of hyaluronan on chondrocyte signaling. Primary chondrocytes were isolated from 6–8 day old BALB/c mice and CD44 knock‐out (KO) mice, as well as adult bovine articular cartilage (CD44 positive). Testicular hyaluronidase (T H'ase) treatment of chondrocytes removed pericellular hyaluronan as visualized by immunostaining. Pre‐treatment with T H'ase stimulated Akt & ERK phosphorylation; this stimulation was muted by CD44 siRNA treatment of bovine chondrocytes. However, pre‐treatment with T H'ase reduced Smad1 phosphorylation and Id2 protein stimulation in response to BMP‐7 in all three chondrocyte models. Thus, in mouse chondrocytes, removal of hyaluronan has a profound effect on cell signaling pathways regardless of endogenous CD44 expression. Receptor compensation may occur in the CD44 KO chondrocytes or alternative mechanisms of hyaluronan‐cell interaction may influence these signaling pathways. Supported by NIH R01‐AR39507 and R01‐AR43384.

Mechanisms of fluid-flow-induced matrix production in bone tissue engineering

Matrix production by tissue-engineered bone is enhanced when the growing tissue is subjected to mechanical forces and/or fluid flow in bioreactor culture. Cells deposit collagen and mineral, depending upon the mechanical loading that they receive. However, the molecular mechanisms of flow-induced signal transduction in bone are poorly understood. The hyaluronan (HA) glycocalyx has been proposed as a potential mediator of mechanical forces in bone. Using a parallel-plate flow chamber the effects of removal of HA on flow-induced collagen production and NF-kappaB activation in MLO-A5 osteoid osteocytes were investigated. Short periods of fluid flow significantly increased collagen production and induced translocation of the NF-kappaB subunit p65 to the cell's nuclei in 65 per cent of the cell population. Enzymatic removal of the HA coat and antibody blocking of CD44 (a transmembrane protein that binds to HA) eliminated the fluid-flow-induced increase in collagen production but had no effect on the translocation of p65. HA and CD44 appear to play roles in transducing the flow signals that modulate collagen production over long-term culture but not in the short-term flow-induced activation of NF-kappaB, implying that multiple signalling events are initiated from the commencement of flow. Understanding the mechanotransduction events that enable fluid flow to stimulate bone matrix production will allow the optimization of bioreactor design and flow profiles for bone tissue engineering.

A role for the endothelial glycosaminoglycan hyaluronan in neutrophil recruitment by endothelial cells cultured for prolonged periods

Glycosaminoglycans (GAGs) presented on the surface of endothelial cells (ECs) are believed to influence leukocyte recruitment during inflammation, but their roles remain uncertain. Here we report an in vitro model of prolonged culture of human EC in which the contributions of heparan sulphate (HS) and hyaluronan (HA) to the process of neutrophil recruitment could be studied. Previously, we reported that increasing EC culture duration (up to 20 days) enhanced neutrophil recruitment in response to low dose (1 U/ml) but not high dose (100 U/ml) of tumour necrosis factor-α (TNF). Here we found that HS and HA were present at much higher levels on the surface of day 20 cultures than day 3 cultures. Neutrophil recruitment on both day 3 and day 20 ECs was mediated through CXCR chemokine receptors and interleukin-8 (IL-8). In addition, mRNA levels for TNF receptors, signalling pathway constituents, adhesion receptors, and chemokines involved in neutrophil recruitment were similar for day 3 and day 20 ECs. To test whether the enhanced neutrophil recruitment on day 20 EC was mediated by GAGs, they were removed enzymatically. Removal of HA (but not HS) inhibited neutrophil recruitment, as did antibody blockade of CD44, a counter-receptor for HA on neutrophils. Supernatants from hyaluronidase-treated day 20 ECs were more potent in activating neutrophils than supernatants from untreated EC. Thus, HA has a role in neutrophil recruitment that is revealed in long-term cultures where it increases potency of response to sub-optimal levels of TNF. This effect appears to occur through a dual mechanism involving chemokine presentation and interaction with CD44.

Enzymatic removal of hyaluronan affects routing of axons in the mouse optic chiasm

Perturbations of interaction of hyaluronan (HA) with its receptor CD44 cause multiple errors in axon routing at the mouse optic chiasm. To investigate this interaction further on the chiasm routing, we studied the axon routing after enzymatic removal of HA from slice preparations of the optic pathway. Hyaluronidase treatment produced an obvious reduction in midline crossing of the first generated axons in E13 chiasms, but had no influence on routing ofthe uncrossed axons in E15 and E16 slices. These findings support a direct role of HA, acting probably through CD44, on axon decussation during early phase of chiasm development, but argue against a direct function of HA on the turning of uncrossed axons in the mouse optic chiasm.

Inducible Hyaluronan Production Reveals Differential Effects on Prostate Tumor Cell Growth and Tumor Angiogenesis

Prostate cancer progression can be predicted in human tumor biopsies by abundant hyaluronan (HA) and its processing enzyme, the hyaluronidase HYAL1. Accumulation of HA is dictated by the balance between expression levels of HA synthases, the enzymes that produce HA polymers, and hyaluronidases, which process polymers to oligosaccharides. Aggressive prostate tumor cells express 20-fold higher levels of the hyaluronan synthase HAS3, but the mechanistic relevance of this correlation has not been determined. We stably overexpressed HAS3 in prostate tumor cells. Adhesion to extracellular matrix and cellular growth kinetics in vitro were significantly reduced. Slow growth in culture was restored either by exogenous addition of hyaluronidase or by stable HYAL1 coexpression. Coexpression did not improve comparably slow growth in mice, however, suggesting that excess hyaluronan production by HAS3 may alter the balance required for induced tumor growth. To address this, we used a tetracycline-inducible HAS3 expression system in which hyaluronan production could be experimentally controlled. Adjusting temporal parameters of hyaluronan production directly affected growth rate of the cells. Relief from growth suppression in vitro but not in vivo by enzymatic removal of HA effectively uncoupled the respective roles of hyaluronan in growth and angiogenesis, suggesting that growth mediation is less critical to establishment of the tumor than early vascular development. Collectively results also imply that HA processing by elevated HYAL1 expression in invasive prostate cancer is a requirement for progression.

A Cost-Effective Method for the Optical Transduction of Chemical Reactions. Application to Hyaluronidase Inhibitor Screening with Polyarginine−Counteranion Complexes in Lipid Bilayers

We report the systematic elaboration of a cost-effective, interference-minimized assay for the label-free optical transduction of chemical reactions. Recently, we have found that certain complexes formed by arginine-rich cell-penetrating peptides (CPPs) and amphiphilic counteranions can act as synergistic anion carriers in lipid bilayer membranes. Application of this discovery to rapid and reversible cytosolic CPP delivery has been described (Futaki, S.; et al. ACS Chem. Biol. 2006, 1, 299). Here, we report the complementary use of polyarginine (pR)-counteranion complexes as general optical transducers of chemical reactions. Counterion screening revealed dodecyl phosphate (DP) as an ideal pR activator. Carboxyfluorescein (CF)-loaded vesicles with a shelf life of 3.5 years served best for the detection of fluorogenic CF release by pR-DP complexes with the naked eye. Inactivation of pR-DP complexes by counterion exchange with hyaluronan (HA) caused no CF emission, while HA removal by hyaluronidase (HAase) did. pR-DP complexes were further compatible with the optical detection of HA immobilization on solid support as well as inhibitor screening for HAase (cromolyn, heparin) with and without substrate immobilization. Controls concerning binary ATP/ADP discrimination for naked-eye kinase detection are mentioned to delineate scope but also limitations of this simple and quite universal method.

Depolymerized Hyaluronan Induces Vascular Endothelial Growth Factor, a Negative Regulator of Developmental Epithelial-to-Mesenchymal Transformation

Cardiac malformations constitute the most common birth defects, of which heart septal and valve defects are the most frequent forms diagnosed in infancy. These cardiac structures arise from the endocardial cushions through dynamic interactions between cells and the extracellular matrix (cardiac jelly). Targeted deletion of the hyaluronan synthase-2 (Has2) gene in mice results in an absence of hyaluronan (HA), cardiac jelly, and endocardial cushions, a loss of vascular integrity, and death at embryonic day 9.5. Despite the requirements for Has2 and its product, HA, in the developing heart, little is known about the normal processing and removal of HA during development. Cell culture studies show that HA obtains new bioactivity after depolymerization into small oligosaccharides. We previously showed reduction in Has2 expression and diminished presence of HA at later stages of heart development as tissue remodeling formed the leaflets of the cardiac valves. Here we show that small oligosaccharide forms of HA (o-HA) act antagonistically to developmental epithelial-to-mesenchymal transformation (EMT), which is required to generate the progenitor cells that populate the endocardial cushions. We further show that o-HA induces vascular endothelial growth factor (VEGF), which acts as a negative regulator of EMT. This is the first report illustrating a functional link between oligosaccharide HA and VEGF. Collectively, our data indicate that following endocardial cell EMT, native HA is likely processed to o-HA, which stimulates VEGF activity to attenuate cardiac developmental EMT.

A Role for Hyaluronan in the Preservation of Interstitial Structure

Hydraulic resistance of interstitium is of major importance in body fluid distribution. In the synovial lining it is vital for the retention of intra-articular fluid, and is attributed chiefly to the network of interstitial biopolymers occupying intercellular gaps in the tissue. Selective removal of synovial hyaluronan (HA) by protease-free hyaluronate lyase results in an almost 10x increase in synovial hydraulic permeability from 0.48 +/- 0.24 microL min(-1) cm H2O (control) to 4.56 +/- 0.40 microL min(-1) cm H2O (mean +/- SD, n = 6 rabbits, p < .001, t test) leading to the hypothesis that hyaluronan plays a major role in the organization of interstitial matrix structure. To test whether removal of hyaluronan causes significant changes in synovial ultrastructure, morphometry of hyaluronidase-treated synovium was carried out. Following hyaluronidase, the thickness of the synovial lining was reduced from 13.0 +/- 1.6 microm (control) to 10.6 +/- 1.6 microm (mean +/- SD throughout, n = 50 measurements per rabbit, 6 rabbits. p < .001, t test). This was accompanied by a significant reduction of synovial interstitial volume fraction from 76.2 +/- 20.6% (control) to 67.04 +/- 24.94% (p < .001, t test), and an increase in collagen bundle volume as a fraction of interstitial volume from 40.75 +/- 4.97% (control) tissue to 48.77 +/- 11.72% (p < .0001, t test). The findings indicate that the removal of hyaluronan chains leads to morphological disruption. Thus, hyaluronan chains play a major role in the organization of synovial structure. The observed morphological changes are insufficiently large to explain fully the great rise in hydraulic permeability observed on HA removal. The latter is likely to be due to disruption of tertiary architecture at the molecular organization level.

Endothelial adhesion of synchronized gastric tumor cells changes during cell cycle transit and correlates with the expression level of CD44 splice variants

To study adhesion capacity and CD44 expression of human gastric adenocarcinoma MKN45 cells at different stages of a first cell cycle. MKN45 cells were synchronized by aphidicolin and assayed for adhesion to an endothelial cell (HUVEC) monolayer. Surface expression of CD44 and CD44 splice variants on MKN45 cells was evaluated by flow cytometry. Functional relevance of CD44 adhesion receptors was investigated by blocking studies using anti CD44 monoclonal antibodies or by hyaluronan digestion. Adhesion of MKN45 to HUVEC was increased during G2/M transit, after which adhesion returned to baseline levels with cell cycle completion. In parallel, CD44 splice variants CD44v4, CD44v5, and CD44v7 were all up-regulated on MKN45 during cell cycle progression with a maximum effect in G2/M. The function of CD44 surface receptors was assessed with specific receptor blocking monoclonal antibodies or removal of hyaluronan by digestion with hyaluronidase. Both strategies inhibited tumor cell adhesion to HUVEC by nearly 50%, which indicates that MKN45-HUVEC-interaction is CD44 dependent. CD44 expression level is linked to the cell cycle in gastrointestinal tumor cells, which in turn leads to cell cycle dependent alterations of their adhesion behaviour to endothelium.

Bone Morphogenic Protein-7 Inhibits Monocyte-Stimulated TGF-β1 Generation in Renal Proximal Tubular Epithelial Cells

It has been demonstrated that bone morphogenic protein-7 (BMP-7) stimulates formation of hyaluronan (HA)-based cables on the cell surface of renal proximal tubular cells and that these cables mediate monocyte binding. Furthermore, interaction of monocytes with proximal tubule cell (PTC) surface intracellular adhesion molecule (ICAM) stimulates the synthesis of TGF-beta1. This study examined the effect of BMP-7 on monocyte-stimulated TGF-beta1 synthesis under conditions of basal and stimulated ICAM expression. Monocyte (U937 cells)-dependent stimulation of TGF-beta1 promoter activity and protein synthesis was reduced by addition of BMP-7 for 24 h before addition of U937 cells. Removal of cell surface HA or inhibition of monocyte interaction with HA using antibody to CD44 prevented this effect of BMP-7. These data suggest that BMP-7 enhances HA-dependent binding and reduces ICAM-dependent binding, which is known to stimulate TGF-beta1 synthesis. This hypothesis was examined further by stimulation of PTC ICAM expression by TNF-alpha. After TNF-alpha stimulation, monocyte-dependent TGF-beta1 synthesis increased. This was abrogated by inhibition of ICAM-CD18 interactions. TNF-alpha stimulation alone did not increase TGF-beta1 synthesis. TNF-alpha stimulation of PTC in the presence of BMP-7 failed to increase monocyte-dependent TGF-beta1 stimulation. Although stimulation of PTC by BMP-7 alone decreased cell surface ICAM expression, it did not affect TNF-alpha-induced ICAM expression. The effect of BMP-7 on TGF-beta1 synthesis in TNF-alpha-stimulated cells was abrogated by disruption of CD44-HA interactions, suggesting that it was due to increased monocyte binding to HA on the cell surface.

Hyaluronan mediates adhesion of metastatic colon carcinoma cells 1

Hyaluronan (HA) is a cell-surface glycosaminoglycan that has been implicated in cancer progression. Cells isolated from metastatic colon carcinoma (SW620) produce greater amounts of pericellular HA than cells isolated from a primary tumor (SW480). Inhibition of hyaluronan synthases (HAS) by transfection with antisense cDNA decreases HA production. Because adhesion to the extracellular matrix (ECM) is required for invasion and metastasis, we hypothesized that pericellular HA mediates adhesion to ECM proteins such as laminin, collagen, and fibronectin and that inhibition of HA production or removal of HA by digestion with hyaluronidase would impair adhesion. SW480, SW620, and antisense transfectants (SW620 cells transfected with vector alone, antisense HAS2, antisense HAS3, and both antisense HAS2 and HAS3) were assessed for adhesion to laminin, Type 1 collagen, or fibronectin-coated plates. To confirm that adhesion was mediated by HA, cells were treated with or without hyaluronidase prior to the assays. Metastatic SW620 cells adhered well to laminin; SW480 cells demonstrated 46% less adhesion (P < 0.05; Student's t test). SW620 cell adhesion to Type 1 collagen and fibronectin was >50% less than adhesion to laminin. Inhibition of HAS2 and/or HAS3 or pretreatment with hyaluronidase significantly decreased adhesion of SW620 cells to laminin (P < 0.05), suggesting that adhesion was dependent upon pericellular HA. Metastatic SW620 cells that produce large amounts of pericellular HA adhered well to laminin. Inhibition of HAS2 and/or HAS3 expression, or hyaluronidase digestion of pericellular HA significantly inhibited adhesion. These data suggest that HA promotes adhesion to laminin and may thereby facilitate invasion of the basement membrane and metastasis in colon carcinoma.

Renal Proximal Tubular Epithelial Cell Transforming Growth Factor-β1 Generation and Monocyte Binding

With increasing awareness of the importance of renal cortical interstitial fibrosis, interest has focused on the mechanisms that stimulate generation of profibrotic factors including transforming growth factor (TGF)-beta1, by resident cells, such as proximal tubular epithelial cells (PTCs). Infiltration of monocytes, has been implicated in the pathogenesis of a wide variety of renal diseases, however, how interaction between monocytes and PTCs may affect the generation of TGF-beta1 by the resident cell is unknown. We demonstrate that monocytes stimulate TGF-beta1 transcription and protein synthesis by PTCs. This was dependent on direct cell contact and TGF-beta1 transcriptional activation that was dependent on ICAM-1 binding of unstimulated monocytes. This was mimicked by antibody cross-linking of PTC surface ICAM-1. We have previously identified hyaluronan (HA)-based structures on the surface of PTCs, both primary cultures and the HK-2 cell line. Removal of cell-surface HA increased ICAM-1-dependent monocyte binding and stimulation of TGF-beta1 synthesis. Furthermore, we demonstrate that binding of monocytes to HA-based structures on the cell surface of HK-2 cells interferes with this response. In summary, we have demonstrated that HA-based pericellular structures down-regulate proinflammatory and profibrotic responses by modulation of monocyte-driven ICAM-1-dependent cell activation and TGF-beta1 generation.

Cell‐surface glycosaminoglycans inhibit cation‐mediated gene transfer

Cationic polymers and liposomes are used to wrap DNA into complexes that promote its cellular uptake. The mechanisms of the uptake and the intracellular fate of these complexes are obscure, as are reasons for an unpredictable and sometimes poor efficiency of the transgene expression. Polyanionic glycosaminoglycans (GAGs) on the cell surface interact with the cationic DNA complexes and influence transfection. The quantities of heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronan (HA) on the cell surface of mutated Chinese hamster ovary (CHO) cells and manipulated (chlorate, xyloside, chondroitinase ABC or Streptomyces hyaluronidase) smooth muscle cells were correlated with the uptake of four different DNA complexes, and the expression of the transgene. Two CHO mutants, without cell-surface HS and CS, showed a 1.5-6-fold increase in cellular association of DNA, and 3-25-fold increase of transgene expression, as compared with the wild type. A CHO mutant with a 5.7-fold increase of cell-surface CS, but devoid of HS, showed enhanced DNA association, but 20-40% reduction in its expression. The removal of HS, CS, or HA from the cell surface of smooth muscle cells had a minor or insignificant effect on the cell association and transfection of the carriers and only polyethyleneimine showed increased association and expression of the transgene. The uptake of DNA complexes varies depending on carrier, cell type and amounts of cell-surface HS, CS, and HA, whereas all GAGs inhibit the transgene expression. This implies that cell-surface GAGs probably direct complexes into intracellular compartments that do not support transcription.

375 Gene expression analysis of galactosamine-induced hepatotoxicity in-vivo and in-vitro

Plasma hyaluronan (HA) concentration and the rate of HA uptake by the isolated, perfused liver were measured in rats treated with saline, D-galactosamine (GaINH2, 50 mg/100 g body wt), gadolinium chloride (GdCl3) (0.5 mg/100 g body wt), and GdCl3 + GaINH2. GdCl3 was given 24 hr before GaINH2 or saline. Plasma L-alanine:2-oxoglutarate aminotransferase (EC 2.6.1.2), a marker for hepatocyte damage, was increased by 8 hr and remained elevated for 24 hr after GaINH2 injection. GdCl3 did not affect plasma enzyme levels when given alone or in association with, but prior to, GaINH2. Plasma HA levels were increased (200%) within 2 hr GaI administration. A plateau was reached at 8 hr, which was maintained for at least 24 hr. Although GdCl3 alone did not affect plasma HA levels, it slightly delayed the increase in HA concentration in GaINH2-treated rats. Liver, isolated 24 hr after GaINH2 treatment, exhibited a severe depression (approximately 67%) of HA uptake. GdCL3 did not prevent this suppression. The data presented indicate that: (1) one of the sinusoidal endothelial cell-dependent functions of the liver, i.e. removal of HA from the blood stream, is profoundly impaired during galactosamine-induced hepatitis, and (2) the adverse effect of GaINH2 on this sinusoidal endothelial cell function may not be dependent on GdCl3-suppressible Kupffer cell functions.

The Role of Extracellular Matrix in Transperitoneal Transport of Water and Solutes

To define the extracellular matrix (ECM), to discuss the physical properties of its components and their impact on transport, and to review data in humans and in animals on the importance of hyaluronan to peritoneal dialysis. Literature survey. The ECM fills the interstitium between parenchymal cells and blood vessels in the subperitoneal interstitium. It is responsible for the interstitial resistance to solute and water transfer through the peritoneal barrier. Major components are collagen and hyaluronan, which are synthesized locally in the peritoneal tissue. Synthesis and deposition of these components increase with inflammation, and concentrations of the components influence the mechanical properties of the tissue and the interstitial Starling forces as well as transport. Removal of hyaluronan appears to increase the rates of water and large-solute transport. Addition of hyaluronan to dialysate appears to enhance fluid recovery and to reduce protein loss. Many of the physicochemical properties of ECM components are well described, but a large knowledge gap remains concerning the in vivo consequences of specific alterations in the interstitial components. More research is needed.

Fibrotic healing of adult and late gestation fetal wounds correlates with increased hyaluronidase activity and removal of hyaluronan

The lack of scarring and fibrosis in healing fetal skin wounds may relate to a prolonged presence of hyaluronan (HA). It has been suggested that fetal wounds may lack hyaluronidase, but the hyaluronidase levels in fetal wounds remain unknown. The size of HA influences its biological action, especially in relation to angiogenesis, which is also reduced in fetal wound healing. The present study determined the levels and size of HA, as well as hyaluronidase levels, in fetal and adult lamb wounds. Wire mesh cylinders, or polyvinyl acetate sponges, were placed subcutaneously in fetal lambs at 75, 100 or 120 days gestation. Wound fluid and wound tissue were harvested 3, 7 or 14 days later. Samples were digested with papain and both HA and hyaluronidase activity were determined in a competitive ELISA assay. Size distribution of HA was estimated using a Sephacryl S1000 column and fractions were collected for HA determination. Adult wound fluid HA remained low (4–5 μg/ml) over the 14 days. Fetal fluids were similar on day 3, but increased to 15–25 μg/ml by day 7. In 75/100-day wounds, HA remained elevated at 14 days, but in 120-day fluids decreased to levels similar to adult fluid. The HA in all fluids was polydisperse with a main peak at 200 kDa. Hyaluronidase levels were detected in all samples, reaching a peak 7 days post-wounding. In adult wound fluids hyaluronidase was much higher than the fetal wound fluids. These data suggest that lower hyaluronidase levels in fetal wounds may underlie the different pattern of HA deposition seen in fetal wounds.

Effect of increased interstitial fluid flux on fractional catabolic rate of high molecular weight [3H]hyaluronan injected in rabbit skin.

The removal rate of high molecular weight (M(r) 3 x 10(6) [3H]hyaluronan has been measured in rabbit paw skin in control and during increased local venous pressure induced by ligation of the femoral vein. The increased venous pressure resulted in a 20% increase of interstitial volume at 4 h after ligation, a value which relates to at least a doubling of lymph flow, i.e. also to doubling of fluid flux through the tissues. The fractional catabolic rate of hyaluronan was 0.0918 +/- 0.0170 h-1 (n = 10) and 0.0775 +/- 0.0206 h-1 (n = 11) in control and following increased venous pressure respectively (P > 0.05). Since the fractional catabolic rate of hyaluronan is not affected by increased venous pressure, the removal of hyaluronan via lymphatic drainage in rabbit paw skin must be small compared to the local turnover of the polysaccharide.