Koji Kimata
Advanced Medical Research Center,
Aichi Medical University
HA 2013, the 9th International Conference on Hyaluronan Research, was held for six days from June 2 -7, 2013, in Oklahoma City, USA. The Conference was organized mainly by Dr. Paul Weigel, together with four organizers, Dr. Paul DeAngelis, Dr. Anthony Day and preceding organizers Dr. Masaki Yanagishita and Dr. Koji Kimata. It was held in honor of Dr. Bryan Toole for his longtime contribution to hyaluronan (HA) research.
Dr. Paul Weigel addressed concerns over the tornado that struck Oklahoma City immediately before the conference, confirmed that the venue was free from any damage, and the Conference convened as scheduled. There were approximately 240 attendees, and 176 presentations were given (including 13 with no abstracts).
Overall at this 9th Conference the number of reports of discoveries new HA-related molecules decreased markedly compared with previous conference, and papers on newly discovered HA functions and detailed analyses of the functions comprised the majority of the program.
It should be noted that some reports of HA functions vary depending on the chain length. ISHAS has long been aware that this is a problem. The absence of HA standards regarding chain length and molecular weight render comparisons difficult, and the results remain ambiguous.
The program is with a complete list of session titles and summaries of a number of presentations are shown below. These summaries provide the readers some details of the Conference. Photographs from various events are included at the end of this report.
Sophia Khaldoyanidi :
The involvement of its microenvironment is critical to hematopoietic stem cells (HSC) differential regulation in the bone marrow. HSC level was studied by using knockout mice of all three hyaluronan synthase genes (Has1, Has2, Has3). Reduction of HA synthesis by 4MU treatment in culture of bone marrow cells, and effect on HAS differentiation by addition of HA to the culture were also investigated. Observations showed that HA is indeed an important differentiation factor in the structure of hematopoietic microenvironment. Further investigation on its polymer size and conformation may lead to potential clinical application.
Hiroyuki Yoshida :
Normal human skin fibroblasts (Detroit 551 cells) can depolymerize exogenously added HA (> 1,000 kDA) to intermediate-sized fragments (10-100 kDa) in culture. This cell line expresses CD44 and HYAL2 but not HYAL 1. The down-regulation of CD44 and HYAL2 using specific siRNAs showed no definite effect on HA depolymerization.This suggests existence of an unknown HA degrading machinery.
This degrading enzyme activity is significantly up-regulated by histamine treatment and is down-regulated by TGF-β1. Twenty-five genes were found to up- or down-regulate by such treatment. Among them was the KIAA1199 gene, which encodes a deafness gene of unknown function. HA degrading activity in Detroit 551 is inhibited by KIAA1199 siRNA treatment. Furthermore, KIAA1199 cDNA transfection into cells without HA degrading activity specifically upregulated its HA degrading activity. These results indicated that KIAA1199 have specific HA depolymerization activity.
Further investigation revealed that KIAA1199 binds specifically to HA and also immunoprecipitated with the clathrin heavy chain. The siRNA for clathrin heavy chain suppressed the HA depolymerizing activity of KIAA1199 transfected cells, suggesting that KIAA1199 depolymerized HA through the clathrin-coated pit pathway. Moreover, structural analysis of degraded HA showed that, HA depolymerizing activity of KIAA1199 seemed to be beta-end-N-acethylglucosaminidase activity.
It was a fitting presentation in the very first session for the following two reasons. Firstly for the discovery of a new HA degrading pathway, entirely different from the existing HYAL1 and HYAL2 system, and secondly for proving this was the main degrading pathway for skin, cartilage and synovial membrane which are all active tissues in HA metabolism. However, once the KIAA1195 gene transfected cells were disrupted, the HA degrading activity disappeared (same as HYAL2).There are still questions to be solved, e.g., KIAA1195 specific tertiary structure induced by cell surface microenvironment or by interaction with other molecules may be required for expression activity.
It is interesting that the following novel inhibitors which work on this HA degrading pathway were discovered: (1) monensin, a receptor recycling inhibitor, (2) ammonium chloride, an endosomal-lysosomal system acidification inhibitor, (3) dynasor, an inhibitor for GTPase which is involved in division and endocytosis of a newly developed follicle.The behavior of these inhibitors indicate that the newly reported HA degradation occurs in the acidic compartment prior to the endosome-lysosome binding. (This presentation already has been published. Yoshida H et al., Proc Natl Acad Sci U S A. 2013 Apr 2; 110(14):5612-7.)
Ralf Richter :
The forming process of HA centered extracellular matrix tertiary structure was observed by using a matrix model of HA end-grafted to a film (HA brushes; details in Baranova NS et. al., J. Biol. Chem. 286, 25675-86, 2011) under the following conditions: addition of (1) TSG-6 alone, (2) TSG-6 and IαI, and (3) TSG-6, IαI and pentraxin3. In (1), under appropriate concentration, HA cross-linking occurs through HA mediated TSG-6 polymerization, but the formed HA matrix is unstable and ultimately collapses. In (2), TSG-6 functions not only as a stable matrix component but also as an enzyme in transferring the heavy chains from IαI to HA and is involved in raising HA to a higher level. And in (3), the presence of PTX3 has little effect on the HA matrix, but constructs a stable HA structure by conjugating with heavy chains.
What makes this research different is that they used a system that quantitatively analyzes the tertiary formulation of the matrix structure with QCM-D (quartz crystal microbalance dissipation monitoring) and RICM (reflection interference contrast microscopy).
Victor Nizet :
Siglec-9, a member of sialic acid-binding Ig-like lectins (Siglecs), of human leukocytes exists abundantly on the surface of neutrophils. They are known to bind with terminal sialic acid residues on cell surface glycoconjugates and limit baseline neutrophil activation. The presenters discovered that Siglec-9 on neutrophils strongly and specifically binds with capsular HA of human pathogen group A Streptococcus (GAS) in a V-set domain different from the sialic binding domain. The HA binding by neutrophils Siglec-9, with its genomic DNA derived net-like structure characteristic, was thought to be critical to regulating the activation of neutrophils, e.g., neutrophil extracellular traps (NETs) formation in capturing bacteria, pro-inflammatory cytokine release phenomenon and oxidative burst. Therefore, HA of GAS binds to Siglec-9 on the surface of neutrophils and inhibits its immunoregulatory activities.
In this presentation, a part of the immunomodulatory mechanism of one immunomodulatory lectin on neutrophils was revealed to be suppressed by binding to two different glycan chains, one is a GBS produced sialyl-oligosaccharide and the other is HA produced by GAS.
Paul Weigel :
Dr. Weigel's presentation was designed in a question-and-answer format focusing on the following points regarding the use of purified enzymes from Class I (including mammal HAS) Streptococcus equisimilis HAS (SeHAS); 1) the characteristics of intraprotein pore in enzymes, 2) does HAS produce (GlcNAc)n-UDP oligomer first, and then initiate HA synthesis by adding UDP-GlcUA to the reducing terminals of the oligomers?, 3) how the size and polydispersity of HA are determined, and 4) systems with purified enzymes embedded in liposomes have the potential to be an examination model for activated expression of HAS. In that case, how does HA translocate in liposomes? The presentation included the contents of three poster presentations from Dr. Weigel’s group.
The purified SeHAS enzyme added to liposomes containing dye cascade blue (CB) in their lumen is translocated into the liposome membrane. CB efflux from the liposome to the solution proved the existence of an intraprotein pore (pore hypothesis) in the enzyme protein. When the four Cys residues located around the HAS-membrane junction within the UDP-sugar binding sites of the enzyme are modified, the CB efflux is inhibited along with the reduction of enzyme activity. (It is interesting to note that the reduced level of enzyme activity varies depending on the type of modulating drug used and the variation in modifying time; the CB efflux rates grew accordingly slower).This evidence suggests that these four Cys residues are in the inner mouth of the HAS pore and are heavily involved in HA synthesis. Synthesizing HA by adding HA binding substrate to the enzyme-liposome led to HA translocated into the extending liposome. This suggests that HAS itself has the function of HA translocation. Various multiple interactions such as hydrogen bonding, ionic bonding and hydorphobic bonding, between the HA-UDP enzyme protein and purified HA-UDP, within and surrounding the HAS protein pore, generates topological strength of the enzyme protein. This topological force works to retain the HA-UDP inside the cells or release from the cells via the HAS pore and the cell membrane (retain-release model).
The release force may further be influenced by cell movement, HA binding ECM molecules, etc.
As the HA synthesis develops the molecule diameter grows: 8000-fold larger than the HAS protein (<10nm) in diameter. This makes it easy to assume that this is the main release of HA from HAS.
Purified SeHAS is phospholipid-dependent and only requires UDP-sugar substrates and Mg2+ ions to synthesize HA. After a time lag, it can build and synthesize at the reducing end without a primer. Based on previous reports that SeHAS synthesizes chitin-oligomers with only UDP-GlcNAc addition, they hypothesized that (GlcNAc)n-UDP was synthesized and acted as a self-primer followed by UDP-GlcA and UDP-GlcNAc to synthesize HA-UDP. Firstly, using WGA lectin affinity chromatography, they confirmed that the (GlcNAc)n-UDP was synthesized by adding only UDP-GlcNAc.
Alberto Passi and Markku Tammi :
Both presentations were focused on the regulation mechanism of HAS activity. There are several ways to regulate HA synthesis, such as phosphorylation of HAS2 enzyme, reduced concentration of UDP-sugar substrates by drugs, ubiquitination of HAS2, and N-glycosylation influence over intracellular sorting by HAS2 enzyme. The presenters gave a detailed report on the regulation methods of phosphorylation of HAS2 enzyme and covalent modification of O-GlcNAcylation. Phosphorylase treatment clarified the importance of the phosphorylation of enzyme. They predicted that the AMPkinase (AMPk), which is activated when cells are in low-energy stage, phosphorylates the enzyme and blocks HA-synthesis. To prove this hypothesis, they established an in vitro system to observe the covalent modification of enzyme by AMPk, and as expected, the phosphorylation of Thr110 residue of HAS2 by AMPk blocked HA synthesis.
Based on these observations, AMPk effect on enzyme protein covalent modification was studied through an in vitro system which revealed that when HAS2 is phosphorylated at residue Thr 110, its activity is blocked, proving the prediction to be correct. It is known that activation of the glucosamine synthesis pathway increases HA synthesis but this presentation reported that the inhibition of HAS2 O-GlcNAcylation inhibited HA synthesis, while Ser221 O-GlcNAcylation enhanced enzyme activity.
Dr. Tammi investigated the vesicular traffic from the intracellular compartments inside the HAS enzyme to the plasma membrane, which is a HA synthesis site. He and his research group discovered that Rab 10 (one of the Ras-superfamily GTPases) suppresses HA synthesis by increasing retrograde traffic, and concluded that this is critical as a posttranslational adjustment against the cellular environment and metabolic state.
Bruno Flamion :
Dr. Flamion discovered interesting physiological roles of Hyal1 and Hyal2 by analyzing Hyal1 and Hyal2 KO mice. Hyal2 knockout mice showed craniofacial and vertebral abnormalities suggesting the enzyme's role in bone development. Hyal1 or Hyal2 KO mice showed abnormal accumulation of HA in the plasma and lymph nodes. However, the size of the HA molecules in the Hyal2 knockout mice was larger suggesting that this as the cause of the distortion of lymph nodes specific to Hyal2 knockout mice. Particularly interesting was that high molecular weight HA did not accumulate in other tissues of these knockout mice. This suggested that Hyal2 function is related to the basic activity of high molecular weight HA clearance specific to the lymph node. Hyal2 knockout mice suffered from schistocytic hemolysis accompanied by splenomegaly and thrombocytopenia. Because Hyal2 was expressed throughout the entire endothelium, this indicated microangiopathy. Increase in the circulating levels of endothelial injury markers such as ICAM-1 confirmed this finding. These observations indicated that Hyal2 protects the endothelium against thrombotic stress.
Hyal1, on the other hand, appears to be involved in endothelial dysfunction. This circulating enzyme is continuously captured by endothelial cells and is thought to be largely involved in diabetes-induced endothelial damage. Streptozotocin-induced diabetic mice displayed decrease in glycocalyx thickness and endothelium-derived hyperpolarization factor-mediated vasodilation with high levels of plasma Hyal1. In diabetic Hyal1 knockout mice there were no changes in glycocalyx and vasodiliation. Hence, it makes sense that suppressing Hyal1 activity may be an effective approach in preventing diabetic angiopathy. However, Hyal1 knockout mice develop severe osteoporosis with age. The following should be noted when taking the above approach: releasing HA from bones may be important in bone remodeling because osteoclasts express Hyal1, and HA is known to modulate the differentiation and activity of osteoblasts and osteoclasts.
Mark Lauer :
The binding of the heavy chains from inter-alpha-inhibitor (IαI) to HA occurs by adding tumor-necrosis-factor-induced-protein-6 (TSG-6) into the culture medium. The group tested whether this binding enhances leukocyte adhesion to HA cables which are produced in response to polyinosinic-polycytidylic acid (Poly(I:C)) treatment.By using the murine airway smooth muscle (MASM) culture system, they obtained expected results. The results also included unexpected observations such as production of thicker HA cables, increase of HA accumulation in the pericellular matrix, and decrease of isolated HA in the medium, which needed to be addressed. These events were observed only when TSG-6 was added during the Poly(I:C)-induced HA synthesis, and not observed once the HA synthesis was completed. This suggests that the main role of TSG-6 is to induce HA synthesis and increase the accumulation of HA on the cell-surface matrix.
Siro Simizu :
Dr. Simizu described the regulation mechanism of HYAL1 activity by glycosylation. Trp130 residue of this enzyme is C-mannosylate. Substitution from Trp to alanine of this enzyme decreases Hyal1 secretion and its activity. This suggests that C-mannosylation may play an important role in the regulation mechanism of HYAL1. In the process of these experiments, the research group found that HYAL1 is N-glycosylated at Asn99, Asn16 and Asn350. The functional study of N-glycosylation is under way.
Lola Reid :
Various stem cells like bile duct stem cells, hepatocyte stem cells and pancreas stem cells, etc., which make up the liver, biliary duct and pancreas, are found in the biliary tree of the Brunner's gland. They can be isolated by immunoselection and established in culture in Kubota's medium (KM). When KM is used in combination with HA gels under appropriate mechanical loading, it exhibited optimum condition of thickness and elasticity as stem cell niches. If the stem cells are cultured in the defined medium with HA gel, cells can differentiate into appropriate lineage. Clinical trials are ongoing with these hydrogels comprising stem cells being transplanted into the target tissue in order to regenerate the organ.
This session showcased the potential use of HA as an effective substrate for regeneration of various tissues, modified by gelatin crosslinking or binding to sulfate groups.
Melanie Simpson :
The relapse rate of prostate cancer can be predicted accurately by HA accumulation and increase in Hyal1 activation. Dr. Simpson found interesting phenomena related to this molecular mechanism. Overproduction of HA without increasing degrading activity suppresses tumor growth and angiogenesis, decreases cell adhesion and reduces cell motility and proliferation. Details were analyzed and resulted in the development of a small molecule antagonist of Hyal1 which blocks metastasis and a blocking antibody of the lymphatic endothelial HA receptor HARE. The active site of Hyal1 was identified and resulted in the definition of the molecular basis of the metastasis of prostate tumor cells expressing Hyal1.
Paraskevi Heldin :
This research group investigated the role of HAS2 in breast tumor progression by studying the mechanisms through direct signals via its receptor CD44 alone, or via receptors for TGF-β and/or PDGF-BB that cooperate with CD44. IQGAP and iASPP were identified as the proteins which interact with the CD44 cytoplasmic domain by proteomic analysis. Studying IQGAP1/CD44 interactions revealed that HA-induced activation of Rac1 is dependent on IQGAP1, while depletion of IQGAP1 promotes RhoA activation. These observations suggest that CD44 and its complexes formed with these molecules are regulated differently in response to external stimuli from HA, TGF-β and PDGF-BB. The effect on cell proliferation and apoptosis of the interaction between these complexes is yet to be investigated.
Carmela Ricciardelli :
Ovarian cancer is the fourth most common cause of cancer-related death in women. Although removal surgery followed by combined platinum and taxane-based chemotherapy is found effective, over 80% of patients eventually relapse and become resistant to chemotherapy. This chemoresistance can lead to death. Therefore, decreasing the resistivity in recurrent cancer cells has the potential to improve survival.
HA-ECM-CD44 has been shown to play a critical role in ovarian cancer metastasis. This study revealed the association of HA to the development of ovarian cancer chemoresistance. HA treatment reduces effectiveness of carboplatin to induce cell death in cancer cells and upregulates the expression of ABC membrane transporters known to regulate cellular efflux of the drugs. Surprisingly, chemotherapy treatment increases HA production in these cancer cells and HA levels in serum. High serum HA concentration prior to chemotherapy treatment was associated with reduced survival rate. Chemoresistance in cancer cells is thought to be acquired by responding against chemotherapy and involves the increased expression of ABC transporters via a HA-CD44 mediated pathway. Therefore, the HA-CD44 pathway may be a target for overcoming chemoresistancy and improving ovarian cancer survival.
Bryan Toole :
Based on accumulating evidence, invasive tumor cells employ a specialized structure termed invadopodia to break through structural barriers such as basement barriers. Invadopodia is a lipid raft-enriched membrane protrusion containing actin-based membrane-type-1 matrix metalloproteinase (MT1-MMP) and several signaling proteins. CD147 (emmprin; basigin) is a type of immunoglobulin superfamily molecule is known for its strong association with tumor invasion and metastasis, and is also known to induce various synthetic production of matrix metalloproteinases. The highly expressed CD147 on non-neoplastic and non-invasive breast epithelial cells was found to induce MT1-MMP followed by invadopodia-like structure formation, and acquired invasiveness. Moreover, CD147 and MT1-MMP were found in close proximity within the invadopodia-like structures and fractionate in membrane compartments with the properties of lipid rafts. Importantly, CD147 induces HA synthesis and signaling pathways downstream of the HA-receptor.
Recently, Dr. Toole discovered that CD147 induces invadopodia activity and invasiveness by promoting EGFR-Ras-ERK signaling dependent on HA-CD44 interactions. It was also observed that activated Ras and ERK regulates CD147 expression, HA synthesis, and formation of CD147-CD44-EGFR complexes. Malignant breast cancer cells are heterogeneous in their expression of CD147 expression, and highly expressed CD147 are strongly correlated to cell surface EGRF and CD44 levels, activated EGFR and ERK1, and activated invadopodia. From these results, it was concluded that HA-CD44-CD147 interaction is a major factor in regulating cell membrane signaling complexes that are critical for malignant invasion.
The majority of the presentations were given in this session, and studies on the involvement of HA in development, proliferation and metastasis of cancer are expected to continue to increase.
This was the first time a workshop was organized. Educational lectures were given by experienced veterans for young researchers interested in HA research and others who have come to understand the involvement of HA. Topics included the following basic HA analysis methods:
1) assay methods
2) isolation methods
3) measuring methods for purity in obtained HA preparations
4) HA chain length (size) analysis methods
A number of take-home messages were included in the workshop, e.g., commercially available Streptococcus hyaluronidase may contain EGF, TAE (tris-acetate-EDTA) or TBE (tris-borate-EDTA) should be used for agarose electrophoresis of high molecular weight HA, DEM (disposable electrochemical microsensor) is recommended for analyzing small amounts of HA below 0.1μg/ml, etc.
Tribute :
Four preeminent HA scientists, Drs. John Scott (UK), John Sheehan (UK), Robert Fraser (Australia), and Dick Heinegard (Sweden) passed away during 2010 to 2013. Researchers closely associated with them presented slides of their achievements and paid tribute to the leaders in this field.
Warren Knudson :
The loss of HA in the cartilage accompanied by the loss of aggrecan is one of the major development factors in arthritis. The overall level of HA within cartilage ECM is regulated by HA synthase activity and CD44-mediated pericellular HA degradation. Interestingly, human chondrocytes specifically express spliced variant of CD44 (CD44exon 19, increases when cartilage is exposed to IL-1). Specific knock down of this CD44 variant resulted in enhanced HA endocytosis via conventional CD44 (CD44wt, CD44 exon20). This suggests that CD44exon19 acts as a CD44 dominant negative (CD44-DN) to contribute to the functional recovery of cartilage by suppressing CD44-mediated HA endocytosis in arthritis. First, CD44-DN was transduced into the bovine articular cartilage by adenovirus and the expression confirmed by western blotting. Higher increase in CD44-DN expression resulted in higher HA secretion into the medium. Next, an attempt was made to enhance HA retention capacity in tissues by transducing versican G1 (Ad-vG1) via adenovirus. The 40 kDa vG1 domain continually synthesized bound to HA, and resulted in the upregulation of HA holding capability. These results indicate that naturally expressed CD44-DN in cartilage is involved in increasing HA levels by blocking HA endocytosis.
Suneel Apte :
Versican is a chondroitin sulfate proteglycan that aggregates with HA and forms the ECM in most connective tissues. It also binds to fibrillin-1 and fibulin via its G3 domain. Secreted metalloproteinases ADAMTS 1, 4, 5, 9 and 20 specifically cleave the versican at two sites; Glu441-Ala442 of the DPEAAE441-ARR sequence of the GAGβ domain, and Glu405-Gln406 of the NIVSFE405-NQKAT sequence of the GAGα domain. Neoepitope antibodies against the cleaved sequence were established. These antibodies helped to understand that versican degradation occurs in the morphogenetic process of various organs and tissues. In other words, the existing form of HA changed with versican degradation. It was also discovered that the S527A sequence adjacent to the GAG binding site plays a significant role in ADAMTS-5 activity.
Anna Plaas :
Three HA synthases (Has1, 2 and 3) express in the cartilage, and while the importance of HAS2 has been presented, the roles of HAS1 and HAS3 were not clear. Has1-/-, Has3-/-, and Has1,3-/- mice were subjected to unilateral damage of the femoral groove cartilage. Has1-/- and Has1,3-/- showed no signs of cartilage repair suggesting that HAS1 is critical in cartilage regeneration. On the other hand, accelerated cartilage regeneration in Has3-/- KO mice leads to the conclusion that HAS3 appears to suppress cartilage repair. Results with HA staining also supported these speculations.
The two short talks which followed stressed the importance of HA degradation by Hyal1 or high-molecular-weight HA in bone metabolism.
The poster presentation related to these topics showed the following.Since the lubricant function was required in the layer between muscle and fascia for smooth contraction and relaxation, the fibroblast-like cells (fasciacytes) and HA accumulation were definitely observed in this area. Noteworthy was the importance of HA not only in muscle differentiation but its involvement in muscle function and maintenance.
From eight 10-minute poster presentations, it was evident that HA is now well on its way to medical use.
Sema Kaderli reported the effect of anti-oxidants, such as 2AP and 4ASA, covalently bound to HA by EDC in OA treatment. Fanny Longin presented the development of a drug delivery system using HA hydrogels cross-linked with divinyl sulfone which allowed the control of sustained drug release and delivery to the affected area. Daniela Smejkalova synthesized acylated HA derivatives of varying length, from C6 to C18, and examined the effect on self-assembly in aqueous solutions.
Jens Fischer :
Since HA and versican-rich ECM are one of the characteristics of occlusive thrombus in arteriosclerosis, which means that promoting HA synthesis promotes thrombus, it was thought that augmentation of HA synthesis promoted thrombus and that the inhibition of HA synthesis prevented it. However, in ApoE-deficient mice, HA synthesis inhibition with 4MU resulted in endothelial glycocalyx decrease and acceleration of thrombus and inflammation. Based on these findings, it seems to be important to specifically inhibit HA synthesis in the vascular smooth muscle cell (VSMC) and leave the endothelial glycocalyx intact in atherosclerosis treatment.
In the thrombus of ApoE-deficient mice, HA accumulation caused by upregulated HAS3 with machrophage invasion was observed. Further investigation revealed that cytokines such as IL-1β and TNF-α released from activated machrophages specifically upregulated HAS3 expression in VSMC and resulted in HA synthesis induction. It was assumed that focal adhesion kinase (FAK) signaling from CD44 and RHAMM induced VSMC activation and subsequent cell migration and proliferation, followed by monocytes and macrophages adhering to HA-rich EMC, eventually leading to signal enhancement which accelerates thrombus and inflammation. Thus, regulation of HA synthesis via HAS3 will be key to developing anti-atherosclerosis agents.
Paul Bollyky :
Since low molecular weight HA promotes pro-inflammatory responses and tissue regeneration is well known, Dr. Bollyky examined the mechanism of high molecular weight HA (HMW-HA) suppressing inflammation and obtained interesting results. He discovered that HMW-HA promotes the function of regulatory T-cells (Treg) and inhibits the proliferation of conventional T-cells, where CD44, a HMW-HA receptor, suppresses STAT5 phosphorylation, which is critical for Treg survival and the production of anti-inflammatory cytokine IL-10. The same thing occurred by crosslinking of CD44. On the other hand, HA fragments suppressed the crosslinking of CD44 and did not promote STAT5 phosphorylation. This signal pathway appears to be impaired in type 1 diabetes, an autoimmune disease where both Treg and STAT5 signaling are involved in the disease pathogenesis. Thus, it was suggested that HMW-HA creates the effect by regulating STAT5 signaling and sustaining Treg.
Davide Vigetti :
Has2 transcription is regulated by chromatin modulation with epigenetic reaction. In order to determine possible functions modifiable by O-GlcNAc in histones, focus was placed on the chromatin structure around the gene promoter of HAS2 and HAS-AS1, long noncoding RNA with complementary sequence with HAS2 exon 1. Surprisingly, by O-GlcNAcylation chromatin achieved an open state allowing both promoters easy access to the transcription machinery. These new findings on the regulation of HA metabolism may provide new strategies for anti-atherosclerotic and vaso-protective drug development.
Cecile Onclinx :
Dr. Onclinx generated Hyal2-deficient mice, which were believed to play a central role in the catabolism of HA. Surprisingly, they merely displayed phenotypes of skeletal abnormalities and hematologic diseases. HA concentration in the Hyal2-/- mouse plasma was significantly high. Hyal2 is expressed as a membrane protein on the erythrocyte surface, but the erythrocyte in KO mice showed reduced half-life from 25 to 7 days. The absence of Hyal2 resulted in chronic hemolysis and thrombopenia in the presence of schizocytes and endothelial cell abnormalities, but the details of this mechanism remain to be clarified.
Robert Steadman :
Fibroblast to myofibroblast differentiation occurs in the process of tissue repair in organ damage such as found in kidneys. This triggers tissue fibrosis. During the differentiation, myofibroblasts express α-smooth muscle actin through HA increase and TGF-β signaling. Details of this process revealed that the fibroblast differentiation responding to TGF-β1 is dependent on and mediated by HA.
CD44 and epidermal growth factor receptor (EGFR) co-localize in lipid rafts, inducing downstream mitogen-activated protein kinase (MAPK/ERK) and Ca2+/calmodulin kinaseII (CaMKII) activation. Extracellular signal-regulated kinase (ERK) is upstream of CaMKII, and ERK activation is necessary for CaMKII signaling; therefore both kinases are essential for myofibroblast differentiation. Discovering that this signaling pathway acts synergistically with TGF-β1-dependent SMAD-signaling may provide a novel opportunity to develop an intervention method in wound healing and fibrosis.
Yu Yamaguchi :
Dr. Yamaguchi and his group generated the perineuronal specific HAS knockout mice and found that the mice lacking the Has3gene showed the most severe spontaneous epileptic seizures. The hippocampal HA levels were markedly reduced in Has3-/- mice compared to Has1 and Has2 KO mice. The reduction level was 56%, 14% and 25%, respectively. Electrophysiologic observation of Has3-/- KO mouse brain slices revealed spontaneous epileptiform activity in the hippocampal CA1 region of pyramidal neurons. Histological analysis showed that the cell density in the CA1 stratum pyramidale of this mouse significantly increased, and transit of a fluorescent marker through the extracellular space (ECS) was confirmed.
Quantitative determination of ECS volume demonstrated that ECS in the CA1 region was specifically reduced by about 40% in Has3-/- mice. Experimentally, augmentation or reduction of ECS volume blocks or induces epileptiform activity, leading to the conclusion that the reduction in ECS volume is the mechanism of epileptogenesis in Has3-/- mice. These results revealed that HA plays a critical role in ECS volume, and regulating ECS volume with HA opens new avenues for the development of antiepileptogenic therapeutic agents.
Anthony Day:
Dr. Day demonstrated the participation of IαI, TSG-6 and PTX3 (pentraxin3) molecules in cumulus cell matrix formation using various amino acid mutants of TSG-6.
The Ca2+ ion-binding site within the TSG-6 CUB module mediates HC-TSG-6 complex formation, where the Mg2+ ion binding site within to HC contributes HC dimerization. This weak HC-HC interaction may be critical to HC-HA complex formation.
Thomas Wight :
Versican and HA co-localize to extracellular matrix (ECM) fibrils and cables in cultures of vascular smooth muscle cells and lung fibroblasts when stimulation with agonists induces endoplasmic reticulum (ER) stress to these cells. Leukocytes, including eosinophils, monocytes and T-lymphocytes, also bind to EMC fibrils and enhance inflammation. Removing chondroitin sulfate chains of versican or inhibiting versican synthesis with siRNA reduced monocyte binding to the ECM formed by these cells. Producing an ECM enriched with elastic fibers and depleted in HA and wild type versican by V3 overexpression in cultured lung fibroblasts resulted in significant reduction of monocyte binding. When these cells were injected into injured carotid arteries in rabbits, expression of V3 inhibited macrophage and lipid accumulation and greatly reduced vascular inflammation. This observation indicates that versican and HA are both involved in myeloid and lymphoid cell interactions and that they may be target molecules that control the immune response associated with inflammation in vascular disease.
Carol de la Motte :
Human intestinal epithelial cells produce human beta defensin 2 (HβD2) proteins as a mucosal host defense against pathogenic bacteria. Low expression of HβD2 may contribute to chronic inflammatory conditions, such as inflammatory bowel disease (IBD). Interestingly, 35 kDa HA fragments promote HβD2 expression both in vitro and in vivo in a TLR4-dependent manner. In fact, in a colitis mouse model, HA fragments of this size increased resistance against infection by pathogen Salmonella enterica. The higher amount of HA, polydispersing 1000 kDa to 20 kDa, is present in first 3 months postpartum human milk, and 10% to 15% of it is 35kDa. HT29 cells treated with HA purified from human milk increased HβD2 expression and minimized Salmonella enterica s. typhimurium infection. These data suggest that HA in human milk is an important aspect of the mechanism that prevents infection and subsequent inflammation.
Stavros Garantziotis :
Inflammation and airway hyperresponsiveness is the main problem of airway disease. It is characterized by HA accumulation in the bronchi, and the higher amount of accumulation reflects the severity of immune activation and remodeling of the airway. Hence, the research focus is now on HA's mediating role in immune responses and bronchial stenosis, and modification method of HA signaling for therapeutic purposes. Dr. Garantziotis’s group found that short-chain HA increases at the inflammation site of the airway, followed by TLR4 dependent NF-κβ activation, but anti-IαI antibody treatment, TSG-6 deficiency treatment and high molecular weight HA addition can attenuate hyperresponsiveness. They also noted that short-chain HA activates Rho via CD44 signaling and that Rho kinase inhibition reduces hyperresponsiveness caused by short chain HA.
Maria Grandoch :
Dr. Grandoch investigated the involvement of HA in dextran sodium sulfate (DSS) derived inflammatory bowel disease (BD). Since this drug upregulates HAS3 mRNA expression in the colon, HAS3 knockout mice were used, and it was discovered that inflammation was significantly inhibited and that CD45 expressing leukocytes and the infiltration of macrophage had decreased. However, suppression of HA synthesis by 4MU treatment aggravated the inflammation. Therefore, HA synthesis by HAS1 and HAS2 inhibit inflammation while that by HAS3 has a proinflammatory role. This suggests that various types of HAS have different roles in inflammatory bowel disease.
Yoshihiro Nishida :
Dr. Nishida reported that 4-methylumbelliferone (MU) administration inhibits the metastasis of osteosarcoma, chondrosarcoma, Lewis lung cancer cells and breast cancer cells and suggested that the suppression of HA synthesis and reduced pericellular HA matrix caused by 4MU are greatly involved in this phenomenon. Zoledronic acid, a reagent for bone metastatic and radiation therapy, coadministered with 4MU was also found to be effective.
Tracey Brown :
Over 95% of solid cancer displays the activation or over expression of HA receptor CD44. Based on this finding, a HA-derivatised cytotoxic drug that uses CD44 of the tumor cell as a tumor recognition moiety or a high molecular HA (of large volume) drug encapseling a chemotherapeutic drug which allows control of the metabolism has the potential to become a novel anti-cancer agent. A derivative of HA-irinotecan, a quinoline alkaloid which inhibits DNA topoisomerase I, was developed and found to be effective.
Naoki Itano :
HA serves as a microenvironmental signal for recruiting tumor-associated macrophages (TAM), which is key to neovascularization in tumor progression. Overproduction of HA in Has2 transgenic mice resulted in rapid development of breast cancer and angiogenesis, followed by E-cadherin loss and increased nuclear translocation of β-catenin, indicating that an epithelial-mesenchymal transition (EMT) occurred. Rapid TAM migration to the stromal areas of tumors in a manner dependent on a HA-rich tumor matrix was observed. In contrast, when macrophages were removed by liposomal clodronate, tumor angiogenesis and lymphangiogenesis were reduced. To prove HA involvement in TAM action, Has2 null fibroblasts from HAS2 gene knockout mice were inoculated with tumor cells to nude mice. No macrophage recruitment or significant attenuation of tumor angiogenesis and lymphangiogenesis was observed, indicating that a HA-rich stromal microenvironment is critical to tumor neovascularization and tumor progression.
M. Laird Forrest :
HylaPlat, a cisplatin conjugated to HA, was evaluated in canines and rats. Compared to i.v. cisplatin, HylaPlat increased AUC (area under the blood concentration-time curve: used as an index of the rate of drug absorption or bioavailability: an indication of effectiveness of a drug) of cisplatin 5.4-fold and the lymph node concentration 18-fold in canines. The rat model displayed milder damage in liver and less necrosis in the kidneys. These suggest the potential use of HylaPlat as an effective anti-tumor drug.
Lilly Bourguignon :
Bone marrow-derived mesenchymal stem cells (BM-MSCs) are regulated by the HA/CD44-Oct4/Sox2/Nanog-miR-302 signal pathway. The subpopulation of BM-MSCs expressing high level CD44v3 highly expresses stem cell markers, such as transcription factors Oct4, Sox2, and Nanog. HA stimulation promoted both expression and nuclear translocation of stem cell markers of this subpopulation. While it was revealed that upstream promoters containing Oct4/Sox2/Nanog binding sites regulate microRNA-302 (miR-302) expression, it was also found by chromatin immunoprecipitation (ChIP) assays that the upregulation of miR-302 is HA-dependent. BM-MSCs effectively differentiate into various cell lineages including neurons and astrocytes when treated with HA. When these BM-MSCs were infected with lenti virus with an anti-miR-302 inhibitor in order to silence miR-302 expression, expression of epigenetic regulator, AOF2, was enhanced and HA-mediated BM-MSC differentiation also improved. In CD44 knock-out mice, HA did not enhance Oct4, Sox2, and Nanog expression or differentiation.
Stephen Back :
White matter injury caused by ischemic hypoxia results in myelination failure. Damage at premature birth will lead to life-long neurological disabilities. Dr. Back reported that the HA processing by PH20 plays a role in the generation mechanism, showing that the maturation of oligodendrocyte progenitors was blocked by degraded HA.
Lawrence Sherman :
Dr. Sherman's results were similar to those of the research above. The proliferation and differentiation of neural progenitor cells (NPC) must be strictly controlled both during development and in the adult central nervous system (CNS). One of the regulatory factors is a component of the extracellular matrix, and among those, HA is found at high levels in niches of two adult NPCs of the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus.
In the SGZ, where NPCs are involved in neurogenesis required for learning and memory, the disruption of HA and CD44 expressed on NPC leads to abnormal NPC proliferation and delayed differentiation, and these effects were dependent on the HA fragments generated by PH20. For example, chemical brain lesions caused by cancer chemotherapy agents or ethanol provoke increase in PH20 expression and reduction in HA, which eventually leads to learning and memory deficits in mice. Outside of these niches, parenchymal progenitor cells are recruited to the damaged portion. One of these cells, oligodendrocyte progenitor cell (OPC), becomes a myelin-forming oligodendrocyte. Therefore, HA accumulates in the damaged portions and this inhibits the maturation and myelination reaction of the OPC. Recruited OPC express PH20 and HA degraded by this PH20 inhibits maturation and myelination. Also, the inhibition of this degrading activity promotes remyelination. These results indicate that the balance between HA and PH20 is critical for controlling the proliferation and differentiation of NPCs.
David Jackson :
HA is one of the matrix components within lymphatic endothelial cells. LYVE-1, a member of the Link superfamily with 40% sequence similarity to CD44, in lymphatic endothelial cells functions as a key receptor to HA. However, adhesion assays reveal that LYVE-1 binds weakly to HA. Therefore, how, where and when LYVE-1 binds HA in vivo and how this participates in biological functions have yet to be determined. Changing the receptor-ligand avidity, such as by LYVE-1 self-association, surface clustering and exposure to appropriately configured HA complexes, revealed the mechanisms that can modify HA binding. Furthermore, Dr. Jackson showed that by clustering, LYVE-1 selectively binds HA, and HA complexes on the dendritic cells are involved in lymphatic endothelial cell trafficking.
Rosanna Malbran Forteza :
Human airways are normally exposed to bacteria and dust, and the airway epithelium contributes to their clearance. High molecular weight HA (1-1.5 MDa) is synthesized by epithelium in the matrix, binds proteins such as Kallikrein 1(KLK1) and lactoperoxidase, and plays an important role in host defense. TSG-6 produced by epidermal cells interacts with inter alpha trypsin inhibitor (IαI) leaked from the vasculature. This results in the IαI heavy chain transferring to HA and the release of bikunin, which inhibits KLK1. It is speculated that these reactions allow the respiratory epithelia to respond to environmental challenges in lung tissues.
Daniel E. Vaughn :
HA works as a barrier to bulk fluid flow, delaying subcutaneously injected drug absorption. Recombinant human hyaluronidase (rHuPH20) was developed to improve this situation. Clinical studies have demonstrated the possibility of rHuPH20 as a potential agent for facilitating insulin administration.
Glenn Prestwich :
A synthetic extracellular matrix (sECM) gel was created from HA as a biomaterial which will enable the delivery of progenitor cells to appropriate parts, such as skin, liver, heart, brain, bone, cartilage, etc., and aid cell growth and differentiation, in cell therapy. Non-animal derived semi-synthetics sulfated GAGs (SAGEs) were also developed. These proved to be effective as an inhibitor of cationic proteases, P- and L-selectin, and also as an antagonist of RAGE (advanced glycation end-products) receptors. The possibility of treatments for interstitial cystitis and periodontal diseases is also being explored.
Manglio Miguel Rizzo :
Dendritic cells (DCs) are known to play a critical role in immune response. Previous studies suggested that inoculating low molecular weight HA (LMWHA) into mice with colon cancer cells showed an increase in tumor specific immune response. In order to improve the efficiency to produce antibodies against human tumor cells, the possibility of adding LMWHA to a cocktail for DCs maturation was evaluated.
DCs from both healthy volunteers and patients with metastatic colon tumor were stimulated with the cocktail containing LMWHA, and the expression of MHC II, the marker for differentiated DCs, was assessed by flow cytometry. In addition, in vitro and in vivo cell migration capability, metalloproteinase 2 and 9 activity by zymography and changes of HA receptor expressions were also assessed. LMWHA may be useful as an adjuvant in immunotherapy in these cases.
Photo 1. Banquet at the Renaissance Hotel, June 6th.
From left to right : Dr. Bryan Toole, ISHAS President Dr. Paul Weigel and Dr. Warren Knudson.
Photo 2. The banquet featured dances by Native Americans. The music incorporated shades of Japanese folk music known as “minyo”.
Photo 3. Welcome Reception at the Myriad Botanical Gardens, June 3rd.The reception was hosted by organizers Dr. Paul Weigel and Dr. Paul DeAngelis.
From left to right : Dr. Masaki Yanagishita, Dr. Anthony Day, Dr. Vincent Hascall, unidentified guest, Dr. Suneel Apte and the author.
