Hyaluronan Expression Research Articles

Tissue hyaluronan expression, as reflected in the sputum of lung cancer patients, is an indicator of malignancy.

Hyaluronan (HA) shows promise for detecting cancerous change in pleural effusion and urine. However, there is uncertainty about the localization of HA in tumor tissue and its relationship with different histological types and other components of the extracellular matrix, such as angiogenesis. We evaluated the association between HA and degree of malignancy through expression in lung tumor tissue and sputum. Tumoral tissue had significantly increased HA compared to normal tissue. Strong HA staining intensity associated with cancer cells was significant in squamous cell carcinoma compared to adenocarcinoma and large cell carcinoma. A significant direct association was found between tumors with a high percentage of HA and MVD (microvessel density) in tumoral stroma. Similarly significant was the direct association between N1 tumors and high levels of HA in cancer cells. Cox multivariate analysis showed significant association between better survival and low HA. HA increased in sputum from lung cancer patients compared to cancer-free and healthy volunteers and a significant correlation was found between HA in sputum and HA in cancer tissue. Localization of HA in tumor tissue was related to malignancy and reflected in sputum, making this an emerging factor for an important diagnostic procedure in patients suspected to have lung cancer. Further study in additional patients in a randomized prospective trial is required to finalize these results and to validate our quantitative assessment of HA, as well as to couple it to gold standard sputum cytology.

Abstract P5-04-02: Hyaluronan (HA) depletion sensitizes HAhigh tumors to antibody-dependent cell-mediated cytotoxicity

Abstract Therapeutic efficacy of monoclonal antibodies (MAbs) against solid tumor targets, like trastuzumab (anti-HER2) and cetuximab (anti-EGFR), have been used successfully to treat cancer, despite the many physical barriers impeding their access to the malignant cell surface1. For example, only about 50% of HER23+ patients have a durable response to therapy with trastuzumab2. Extracellular matrix (ECM)-mediated inhibition may be among the mechanisms of resistance to MAb therapy of solid tumors. Aberrant accumulation of hyaluronan (HA), a major component of the ECM in many tumors, is associated with poor prognosis and treatment-resistance in multiple malignancies3-5. We investigated HA-dependent pericellular matrix-mediated inhibition to ADCC in HAhigh human cancer cells in vitro and in vivo. We observed high levels of tumor associated HA (HA3+) in >50% of HER23+ breast adenocarcinoma and ∼40% of EGFR+ head and neck squamous cell carcinoma (HNSCC) primary tumors. Human hyaluronan synthase 2 (HAS2)-overexpressing breast cancer cells formed an HAhigh pericellular matrix, which inhibited both natural killer (NK) cell access to tumor cells and ADCC in vitro. Hyaluronan depletion by PEGPH20, a pegylated recombinant human PH20 hyaluronidase currently in clinical study for pancreatic cancer, increased NK cell access to HAS2-overexpressing breast cancer cells and greatly enhanced trastuzumab- or cetuximab-dependent ADCC. Trastuzumab and NK cell accessibility to HAS2-overexpressing tumors was enhanced following HA-depletion by PEGPH20. In an in vivo ADCC-based efficacy study, PEGPH20 treatment in combination with trastuzumab and NK cells enhanced tumor growth inhibition. This work describes a novel tumor microenvironment (TME)-dependent mechanism of inherent resistance to therapeutic antibody-mediated ADCC in vitro and in vivo, and furthermore shows that ADCC can be enhanced by hyaluronan depletion. These results may help to explain as to why tumors with high levels of HA are more aggressive, and suggest potential benefits of PEGPH20-mediated HA depletion in combination with therapeutic antibodies like trastuzumab or cetuximab in the treatment of HAhigh solid tumors.

Hyaluronan expression in primary and secondary brain tumors.

Collectively, primary and secondary brain tumors represent a major public health challenge. Glioblastoma (GBM) is the most common primary brain tumor in adults and is associated with a dismal 5-year survival of only 10%. Breast cancer causes secondary tumors; it occurs in 200,000 patients yearly and 30% of these individuals develop brain metastases which also lead to a very poor prognosis. GBM and primary breast tumors are known to express hyaluronan (HA) which may serve as a therapeutic target. For the present study we had two aims: (I) to identify suitable preclinical models for HA in GBM by examining HA expression in human GBM cell lines implanted orthotopically in mice; and (II) to determine whether breast cancer brain metastases in human patients express HA similar to the primary tumor. Forty human surgical samples of primary breast tumors and breast cancer brain metastases were processed and stained for HA. Athymic nu/nu mice were orthotopically implanted with one of 15 GBM lines and after tumors were established, quantitative immunohistochemistry determined whether. HA was expressed. All GBM cell lines and patient-derived orthotopic tumors did express HA, with 3 primary human lines expressing the highest staining intensity, above that of normal brain. All 40 human primary breast tumors and brain metastases examined also contained HA, though staining intensity was highly variable. Our data support the use of specific patient-derived GBM cell lines in nu/nu mice for preclinical studies on HA-targeting therapies. Additionally, our research provides a basis for the assessment of HA expression and HA-targeting therapeutic agents for the treatment of breast cancer brain metastases.

In vitro 3-D model based on extending time of culture for studying chronological epidermis aging

Skin aging is a complex phenomenon in which several mechanisms operate simultaneously. Among them, intrinsic aging is a time-dependent process, which leads to gradual skin changes affecting its structure and function such as thinning down of both epidermal and dermal compartments and a flattening and fragility of the dermo-epidermal junction. Today, several approaches have been proposed for the generation of aged skin in vitro, including skin explants from aged donors and three-dimensional skin equivalent treated by aging-inducing chemical compounds or engineered with human cells isolated from aged donors.The aim of this study was to develop and validate a new in vitro model of aging based on skin equivalent demonstrating the same phenotypic changes that were observed in chronological aging.By using prolonged culture as a proxy for cellular aging, we extended to 120days the culture time of a skin equivalent model based on collagen–glycosaminoglycan–chitosan porous polymer and engineered with human skin cells from photo-protected sites of young donors. Morphological, immunohistological and ultrastructural analysis at different time points of the culture allowed characterizing the phenotypic changes observed in our model in comparison to samples of non photo-exposed normal human skin from different ages.We firstly confirmed that long-term cultured skin equivalents are still morphologically consistent and functionally active even after 120days of culture. However, similar to in vivo chronological skin aging a significant decrease of the epidermis thickness as well as the number of keratinocyte expressing proliferation marker Ki67 are observed in extended culture time skin equivalent. Epidermal differentiation markers loricrin, filaggrin, involucrin and transglutaminase, also strongly decreased. Ultrastructural analysis of basement membrane showed typical features of aged skin such as duplication of lamina densa and alterations of hemidesmosomes. Moreover, the expression of hyaluronan and its surface receptor CD44 drastically decreased as observed during chronological skin aging. Finally, we found that the level of p16INK4A expression significantly increased supporting cellular senescence process associated to our model.To conclude, the major morphological and ultrastructural epidermal modifications observed in both our extended culture skin equivalent model and skin biopsies from old donors validate the relevance of our model for studying chronological aging, understanding and elucidating age-related modifications of basic skin biological processes. In addition, our model provides a unique tool for identifying new targeted molecules intended at improving the appearance of aging skin.

Antitumor effects of hyaluronan inhibition in desmoid tumors.

Desmoid tumors (DTs) are rare, mesenchymal tumors that exhibit features of an abundant wound healing process. Previously, we showed that mesenchymal stem cells (MSCs) are constituents of DTs and may contribute to desmoid tumorigenesis via activities associated with wound healing. Hyaluronan (HA) is a long-charged chain of repeating glucuronate and N-acetylglucosamine disaccharides that is synthesized by HA synthases (HAS) and degraded by hyaluronidases (HYAL). HA is secreted into the extracellular matrix by injured stroma and is important for normal tissue repair and neoplastic progression. Here, we investigated the presence of HA in DTs and the antitumor effects of the HA inhibitor, 4-methylumbelliferone (4-MU), on DT-derived mesenchymal cells. By immunohistochemistry and enzyme-linked immunosorbent assay, we found abundant expression of HA in 29/30 DTs as well as >5-fold increased HA levels in DT-derived cell lines relative to controls. Immunohistochemistry also demonstrated high expression of HAS2 in DTs, and quantitative PCR analysis showed increased HAS2 upregulation in frozen DTs and DT-derived cells. 4-MU treatment of DT-derived cells significantly decreased proliferation as well as HA and HAS2 levels. Fluorescent immunohistochemistry showed that MSCs in DTs coexpressed HA, HAS2, HYAL2, as well as the major HA receptor CD44 and HA coreceptor TLR4. Taken together, our results suggest that paracrine regulation of HA signaling in DTs may contribute to MSC recruitment and tumor proliferation. Future studies investigating the role of HA in tumor-stroma crosstalk and inhibition of HA-MSC interactions as a novel therapeutic target in DTs and other solid tumors are warranted.

Modifications in bone matrix of estrogen-deficient rats treated with intermittent PTH.

Bone matrix dictates strength, elasticity, and stiffness to the bone. Intermittent parathyroid hormone (iPTH), a bone-forming treatment, is widely used as a therapy for osteoporosis. We investigate whether low doses of intermittent PTH (1-34) change the profile of organic components in the bone matrix after 30 days of treatment. Forty 6-month-old female Wistar rats underwent ovariectomy and after 3 months received low doses of iPTH administered for 30 days: daily at 0.3 µg/kg/day (PTH03) or 5 µg/kg/day (PTH5); or 3 times per week at 0.25 µg/kg/day (PTH025). After euthanasia, distal femora were processed for bone histomorphometry, histochemistry for collagen and glycosaminoglycans, biochemical quantification of sulfated glycosaminoglycans, and hyaluronan by ELISA and TUNEL staining. Whole tibiae were used to estimate the bone mineral density (BMD). Histomorphometric analysis showed that PTH5 increased cancellous bone volume by 6% over vehicle-treated rats. In addition, PTH5 and PTH03 increased cortical thickness by 21% and 20%, respectively. Tibial BMD increased in PTH5-treated rats and this group exhibited lower levels of chondroitin sulfate; on the other hand, hyaluronan expression was increased. Hormonal administration in the PTH5 group led to decreased collagen maturity. Further, TUNEL-positive osteocytes were decreased in the cortical compartment of PTH5 whereas administration of PTH025 increased the osteocyte death. Our findings suggest that daily injections of PTH at low doses alter the pattern of organic components from the bone matrix, favoring the increase of bone mass.

Altered expression of hyaluronan, HAS1-2, and HYAL1-2 in oral lichen planus.

Oral lichen planus (OLP) is an immune-mediated mucosal disease of unclear etiology and of unresolved pathogenesis. Hyaluronan (HA) is an extracellular matrix glycosaminoglycan involved in inflammation and tumor progression. However, its presence in OLP has not been reported. We therefore aimed to study the immunohistochemical expression of HA, its receptor CD44, hyaluronan synthases (HAS1-3), and hyaluronidases (HYAL1-2) in OLP. The presence of HA, CD44, HAS1-3, and HYAL1-2 was studied by immunohistochemical methods in 55 OLP and 23 control oral mucosal specimens (CTR). The localization, intensity, and differences of the epithelial expression between OLP and CTRs were analyzed. HA and CD44 were found on cell membranes in the epithelial basal and intermediate layers in CTR and OLP specimens. The HA staining intensity was stronger in the basal layer of the epithelium in OLP than in CTRs (P < 0.001). HAS1 (P = 0.001) and HAS2 (P < 0.001) showed stronger staining in the basal and weaker staining in the superficial (P < 0.001) epithelial layers in OLP than in CTRs. The immunostaining of HAS3 was low in both OLP and CTRs. Positive HYAL1 and HYAL2 staining were mainly found in the basal and intermediate epithelial layers, and their intensities were significantly increased in OLP, except HYAL 2 in the intermediate epithelial layer. HA, HAS1-2, and HYAL1-2 have altered expression in OLP compared to CTRs and may therefore have a role in OLP pathogenesis.

Reed-Sternberg cell–derived lymphotoxin-α activates endothelial cells to enhance T-cell recruitment in classical Hodgkin lymphoma

AbstractIt is known that cells within the inflammatory background in classical Hodgkin lymphoma (cHL) provide signals essential for the continual survival of the neoplastic Hodgkin and Reed-Sternberg (HRS) cells. However, the mechanisms underlying the recruitment of this inflammatory infiltrate into the involved lymph nodes are less well understood. In this study, we show in vitro that HRS cells secrete lymphotoxin-α (LTα) which acts on endothelial cells to upregulate the expression of adhesion molecules that are important for T cell recruitment. LTα also enhances the expression of hyaluronan which preferentially contributes to the recruitment of CD4+ CD45RA+ naïve T cells under in vitro defined flow conditions. Enhanced expression of LTα in HRS cells and tissue stroma; and hyaluronan on endothelial cells are readily detected in involved lymph nodes from cHL patients. Our study also shows that although NF-κB and AP-1 are involved, the cyclooxygenase (COX) pathway is the dominant regulator of LTα production in HRS cells. Using pharmacological inhibitors, our data suggest that activity of COX1, but not of COX2, directly regulates the expression of nuclear c-Fos in HRS cells. Our findings suggest that HRS cell-derived LTα is an important mediator that contributes to T cell recruitment into lesional lymph nodes in cHL.

Alteration in chondroitin sulfate proteoglycan expression at the epicenter of spinal cord is associated with the loss of behavioral function in Tiptoe walking Yoshimura mice.

The objective of this study was to explore the correlation between the alteration in chondroitin sulfate proteoglycan (CSPG) expression at the epicenter of spinal cord and the loss of behavioral function in tiptoe walking Yoshimura mice. The tiptoe walking Yoshimura mice (twy) and Institute of Cancer Research (ICR) mice, aged 20 and 26weeks, were used in the present study. The behavior assessment, micro-computed tomography and immunofluorescent staining were performed. The compressed spinal cord was histologically analyzed. The results showed that the expression of CSPG was statistically higher at the compressed spinal cord for twy mice compared with that at the normal spinal cord for ICR mice. At the 26th week, a large ossification block at the posterior longitudinal ligament of C1-3 was obviously observed at the micro-CT image We observed the BMS Score was significantly correlated with the expression of glial fibrillary acidic protein, CSPG and hyaluronan (P<0.05). These findings suggest that compression injury induces the higher CSPG expression at the compressed spinal cord in the twy mice. Furthermore, the alteration in CSPG expression at the epicenter of spinal cord is associated with the loss of behavioral function in twy mice.

Divergent Temporal Expression of Hyaluronan Metabolizing Enzymes and Receptors with Craniotomy vs. Controlled-Cortical Impact Injury in Rat Brain: A Pilot Study.

Traumatic brain injury (TBI) triggers many secondary changes in tissue biology, which ultimately determine the extent of injury and clinical outcome. Hyaluronan [hyaluronic acid (HA)] is a protective cementing gel present in the intercellular spaces whose degradation has been reported as a causative factor in tissue damage. Yet little is known about the expression and activities of genes involved in HA catabolism after TBI. Young adult male Sprague-Dawley rats were assigned to three groups: naïve control, craniotomy, and controlled-cortical impact-induced TBI (CCI-TBI). Four animals per group were sacrificed at 4 h, 1, 3, and 7 days post-CCI. The mRNA expression of hyaluronan synthases (HAS1-3), hyaluronidases (enzymes for HA degradation, HYAL 1–4, and PH20), and CD44 and RHAMM (membrane receptors for HA signaling and removal) were determined using real-time PCR. Compared to the naïve controls, expression of HAS1 and HAS2 mRNA, but not HAS3 mRNA increased significantly following craniotomy alone and following CCI with differential kinetics. Expression of HAS2 mRNA increased significantly in the ipsilateral brain at 1 and 3 days post-CCI. HYAL1 mRNA expression also increased significantly in the craniotomy group and in the contralateral CCI at 1 and 3 days post-CCI. CD44 mRNA expression increased significantly in the ipsilateral CCI at 4 h, 1, 3, and 7 days post-CCI (up to 25-fold increase). These data suggest a dynamic regulation and role for HA metabolism in secondary responses to TBI.

Hyaluronan in aged collagen matrix increases prostate epithelial cell proliferation.

The extracellular matrix (ECM) of the prostate, which is comprised primarily of collagen, becomes increasingly disorganized with age, a property that may influence the development of hyperplasia and cancer. Collageous ECM extracted from the tails of aged mice exhibits many characteristics of collagen in aged tissues, including the prostate. When polymerized into a 3-dimensional (3D) gel, these collagen extracts can serve as models for the study of specific cell-ECM interactions. In the present study, we examined the behaviors of human prostatic epithelial cell lines representing normal prostate epithelial cells (PEC), benign prostatic hyperplasia (BPH-1), and adenocarcinoma (LNCaP) cultured in contact with 3D gels made from collagen extracts of young and aged mice. We found that proliferation of PEC, BPH-1, and LNCaP cells were all increased by culture on aged collagen gels relative to young collagen gels. In examining age-associated differences in the composition of the collagen extracts, we found that aged and young collagen had a similar amount of several collagen-associated ECM components, but aged collagen had a much greater content of the glycosaminoglycan hyaluronan (HA) than young collagen. The addition of HA (of similar size and concentration to that found in aged collagen extracts) to cells placed in young collagen elicited significantly increased proliferation in BPH-1 cells, but not in PEC or LNCaP cells, relative to controls not exposed to HA. Of note, histochemical analyses of human prostatic tissues showed significantly higher expression of HA in BPH and prostate cancer stroma relative to stroma of normal prostate. Collectively, these results suggest that changes in ECM involving increased levels of HA contribute to the growth of prostatic epithelium with aging.

Hyaluronan expression as a significant prognostic factor in patients with malignant peripheral nerve sheath tumors.

Hyaluronan (HA) regulates malignant tumor growth, invasion, and metastasis. However, few studies have focused on the roles of HA in tumorigenicity in malignant peripheral nerve sheath tumors (MPNST). In this study, we sought to clarify the prognostic value of HA in patients with MPNST. Specimens obtained from 15 patients with neurofibroma and 30 with MPNST were subjected to HA staining and scored as three grades. Protein expressions of HA synthase 1-3 were examined in the 22 MPNST tissue samples available. Statistically higher HA positivity was observed in MPNST as compared with neurofibroma (P=0.020). The univariate analysis revealed that increased HA expression, age, neurofibromatosis type 1 (NF1) status, large tumor size, and histological grade were significantly associated with reduced overall survival of patients with MPNST; while increased HA expression, NF1 status, tumor size, and histological grade were correlated with disease-free survival. However, HA synthase 1-3 expression related to neither overall survival nor disease-free survival of these patients. In multivariate analysis, large tumor size (P=0.022) was an independent prognostic factor for overall survival, and HA expression (P=0.028) and tumor size (P=0.002) were independent prognostic factors for disease-free survival. Statistically higher levels of HA in the human MPNST cells were observed compared with neurofibroma cells in vitro. Our results demonstrate that HA expression can be a useful marker in differentiating MPNST from neurofibroma, and in identifying patients with a poor prognosis. Hyaluronan-targeting therapy for patients with MPNST may have potential as a therapeutic tool.

Abstract 354: Inducible Disruption of Endothelial Hyaluronan Synthase-2 Expression in Adult Mice Reveals a Direct Involvement of Glomerular Endothelial Surface Hyaluronan in Development of Albuminuria and Renal Injury

We demonstrated that infusion with hyaluronidase of C57Bl/6 mice for 4 wks to chronically reduce EC surface hyaluronan expression resulted in glomerular albumin passage and podocyte injury. However, with systemic hyaluronan degradation it precludes definitive conclusions on cellular-specific effects. Here we investigated the hypothesis that it is specifically the structure and integrity of the glomerular EC glycocalyx that determines whether or not albumin passage across the capillary wall occurs, and subsequently protects against development of kidney injury. To this end, we generated a conditional EC specific hyaluronan synthase2-knock out (HAS2–KO) mouse model. 8 Week old B6.VECad-creERT2.has2f/f.Rosa26-tdTomato were injected i.p. for 5 consecutive days with 2mg/0.2μL tamoxifen (control B6.VECad-creERT2.Rosa26-tdTomato) to induce an endothelial specific deletion of has2. Compared to ctrl, these mice show that 4 wks after induction, weight gain was significantly more increased (mean ± SEM; 2.9 ± 0.7 [n=8] vs. 5.2 ± 0.6g [n=11], p=0.02). With prominent endogenous albumin passage over the barrier, visualized as a DAB reaction product in electron microscopy after anti-albumin-HRP antibody incubation of 2%PFA fixated tissue slides, visible 2 wks after induction, a significant (p=0.036) increased ACR (36.0 ± 1.9 [n=8] vs. 57.7 ± 7.9 [n=11]) was found only at wk 4. During this period no significant change in BP (85 ± 2/114 ± 2 [n=8] vs. 90 ± 4/119 ± 4mmHg [n=10], diastolic/systolic) or in heart rate (712 ± 14 [n=8] vs. 747 ± 8beats/min [n=10]) was observed. Renal morphology revealed with comparable glomerular surface area (5342 ± 270 [n=3] vs. 5508 ± 250 μm2 [n=6]), significant increase of glomerular mesangial area (20.1 ± 0.9 [n=3]) vs. 38.4 ± 1.2% [n=6], p&lt;0.01) and capillary surface area (16.6 ± 1.0 [n=3]) vs. 22.8 ± 0.7% [n=6], p=0.046). In conclusion, the data so far indicate that an intact glomerular EC-ESL is essential for glomerular integrity and prevention of albumin filtration is Key to preserving local quiescence of mesangial cells and podocytes. The conditional EC specific HAS2–KO mouse seems to be a valid model to assess the role of ESL in glomerular permeability barrier and vascular induced glomerular changes.

Bronchial epithelial injury in the context of alloimmunity promotes lymphocytic bronchiolitis through hyaluronan expression.

Epithelial injury is often detected in lung allografts, however, its relation to rejection pathogenesis is unknown. We hypothesized that sterile epithelial injury can lead to alloimmune activation in the lung. We performed adoptive transfer of mismatched splenocytes into recombinant activating gene 1 (Rag1)-deficient mice to induce an alloimmune status and then exposed these mice to naphthalene to induce sterile epithelial injury. We evaluated lungs for presence of alloimmune lung injury, endoplasmic reticulum (ER) stress, and hyaluronan expression, examined the effect of ER stress induction on hyaluronan expression and lymphocyte trapping by bronchial epithelia in vitro, and examined airways from patients with bronchiolitis obliterans syndrome and normal controls histologically. We found that Rag1-deficient mice that received mismatched splenocytes and naphthalene injection displayed bronchial epithelial ER stress, peribronchial hyaluronan expression, and lymphocytic bronchitis. Bronchial epithelial ER stress led to the expression of lymphocyte-trapping hyaluronan cables in vitro. Blockade of hyaluronan binding ameliorated naphthalene-induced lymphocytic bronchitis. ER stress was present histologically in >40% of bronchial epithelia of BOS patients and associated with subepithelial hyaluronan deposition. We conclude that sterile bronchial epithelial injury in the context of alloimmunity can lead to sustained ER stress and promote allograft rejection through hyaluronan expression.

Opposite role of CD44-standard and CD44-variant-3 in tubular injury and development of renal fibrosis during chronic obstructive nephropathy

Chronic kidney diseases (CKDs) are characterized by tubular atrophy and interstitial fibrosis. We previously showed that in obstructive nephropathy de novo CD44 renal expression contributes to renal fibrosis but attenuates tubular damage/apoptosis. As CD44-standard (CD44s) has been linked to TGF-β1-mediated actions and CD44-variant-3 (CD44v3) favors HGF-c-Met binding, we compared the functional properties of these CD44 isoforms in the progression of obstructive nephropathy, using specific CD44-variant knockout/knockin mice. The presence of CD44v3 diminished tubular damage during obstructive nephropathy, decreased apoptosis, and increased proliferation of tubular epithelial cells, and prevented renal fibrosis development. In contrast, expression of CD44s led to increased tubular damage and tubular epithelial cell apoptosis, and more renal fibrosis. A relative increase in renal β-catenin expression, HGF production, and HGF/c-Met signaling, together with a relative inhibition of TGF-β1 downstream signaling and TGF-β type I receptor expression, was found in CD44v3 mice compared with CD44s littermates. In line with this, Wnt3a/HGF treatment of tubular cells resulted in higher β-catenin/p-AKT levels in CD44v3(+) tubular epithelial cells, whereas TGF-β1 induced a mild collagen I upregulation in CD44v3(+) mouse embryonic fibroblasts as compared with CD44s(+) cells. Thus, CD44s and CD44v3 exert opposite roles in the progression of obstructive nephropathy, with CD44v3-v10 being the protective isoform that delays evolution of the renal pathology.

Periostin Induces Intracellular Cross-talk between Kinases and Hyaluronan in Atrioventricular Valvulogenesis

Periostin (PN), a novel fasciclin-related matricellular protein, has been implicated in cardiac development and postnatal remodeling, but the mechanism remains unknown. We examined the role of PN in mediating intracellular kinase activation for atrioventricular valve morphogenesis using well defined explant cultures, gene transfection systems, and Western blotting. The results show that valve progenitor (cushion) cells secrete PN into the extracellular matrix, where it can bind to INTEGRINs and activate INTEGRIN/focal adhesion kinase signaling pathways and downstream kinases, PI3K/AKT and ERK. Functional assays with prevalvular progenitor cells showed that activating these signaling pathways promoted adhesion, migration, and anti-apoptosis. Through activation of PI3K/ERK, PN directly enhanced collagen expression. Comparing PN-null to WT mice also revealed that expression of hyaluronan (HA) and activation of hyaluronan synthase-2 (Has2) are also enhanced upon PN/INTEGRIN/focal adhesion kinase-mediated activation of PI3K and/or ERK, an effect confirmed by the reduction of HA synthase-2 in PN-null mice. We also identified in valve progenitor cells a potential autocrine signaling feedback loop between PN and HA through PI3K and/or ERK. Finally, in a three-dimensional assay to simulate normal valve maturation in vitro, PN promoted collagen compaction in a kinase-dependent fashion. In summary, this study provides the first direct evidence that PN can act to stimulate a valvulogenic signaling pathway.

Hyaluronan Contributes to Bronchiolitis Obliterans Syndrome and Stimulates Lung Allograft Rejection through Activation of Innate Immunity

Although innate immunity is increasingly recognized to contribute to lung allograft rejection, the significance of endogenous innate ligands, such as hyaluronan (HA) fragments, in clinical or experimental lung transplantation is uncertain. To determine if HA is associated with clinical bronchiolitis obliterans syndrome (BOS) in lung transplant recipients, and evaluate the effect of low- or high-molecular-weight HA on experimental lung allograft rejection, including dependence on innate signaling pathways or effector cells. HA concentrations were measured in bronchoalveolar lavage and plasma samples from lung recipients with or without established BOS. BOS and normal lung tissues were assessed for HA localization and expression of HA synthases. Murine orthotopic lung recipients with established tolerance were treated with low- or high-molecular-weight HA under varied experimental conditions, including Toll-like receptor (TLR) 2/4 and myeloid differentiation protein 88 deficiency and neutrophil depletion. HA localized within areas of intraluminal small airways fibrosis in BOS lung tissue. Moreover, transcripts for HA synthase enzymes were significantly elevated in BOS versus normal lung tissues and both lavage fluid and plasma HA concentrations were increased in recipients with BOS. Treatment with low-molecular-weight HA abrogated tolerance in murine orthotopic lung recipients in a TLR2/4- and myeloid differentiation protein 88-dependent fashion and drove expansion of alloantigen-specific T lymphocytes. Additionally, TLR-dependent signals stimulated neutrophilia that promoted rejection. In contrast, high-molecular-weight HA attenuated basal allograft inflammation. These data suggest that accumulation of HA could contribute to BOS by directly activating innate immune signaling pathways that promote allograft rejection and neutrophilia.

Prognostic Impact of Hyaluronan and Its Regulators in Pancreatic Ductal Adenocarcinoma

BackgroundAlthough pancreatic ductal adenocarcinoma is characterized by an abundant stroma enriched with hyaluronan (HA), the prognostic impact of HA and its regulators remains unknown.MethodsUsing immunohistochemistry, expression patterns of HA and its regulators, including a synthesizing enzyme (HAS2), and a degrading enzyme (HYAL1) were investigated in patients who received surgical resection. The prognostic significance of these markers and other clinicopathological variables was determined using univariate and multivariate analyses. The HA levels were determined quantitatively by enzyme-linked immunosorbent assay (ELISA).ResultsWe found that strong expressions of HA (P=0.008) and HAS2 (P=0.022) were significantly associated with shorter survival time after surgery. By contrast, weak expression of HYAL1 was significantly associated with poor survival (P=0.001). In multivariate analysis, tumor stage (hazard ratio (HR)=2.76, 95% confidence interval (CI): 1.14-6.66 P=0.024), strong HA expression (HR=6.04, 95%CI: 1.42-25.69 P=0.015), and weak HYAL1 expression (HR=3.16, 95%CI: 1.19-8.40 P=0.021) were independent factors predicting poor survival. ELISA revealed higher concentration of HA in pancreatic cancer tissues than in normal pancreatic tissues (P=0.001).ConclusionThese findings suggest, for the first time, that HA and its regulators may have prognostic impact in patients with pancreatic cancer.

BMP-2 Induces Versican and Hyaluronan That Contribute to Post-EMT AV Cushion Cell Migration

Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV) valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM) components, versican and hyaluronan (HA), and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH) stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC) aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of versican and Has2 by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of versican and Has2. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions.

Abstract B107: STAT3-induced production of hyaluronan contributes to FGFR-induced migration, proliferation, and therapeutic resistance in breast cancer

Abstract Aberrant activation of fibroblast growth factor receptors (FGFRs) has been linked to breast cancer growth and progression. FGFR activation in tumor cells acts through both autocrine and paracrine mechanisms to contribute to tumor growth. Our previous studies suggesting that FGFR activation leads to increased inflammation led us to evaluate the mechanisms that drive FGFR-induced inflammation in the tumor setting. We demonstrate here that FGFR activation leads to phosphorylation of STAT3Tyr705 in a number of breast cancer cell lines. Furthermore, we demonstrate that phosphorylation of STAT3Tyr705 is mediated indirectly through FGFR-induced production of IL-6 family cytokines. Using a novel in vivo model of FGFR-dependent tumor growth, we demonstrate that pharmacological inhibition of STAT3 leads to decreased FGFR tumor growth. Further examination of the function of STAT3 in this model revealed that STAT3 regulates expression of hyaluronan synthase 2 (Has2) and subsequent production of the extracellular matrix component hyaluronan (HA). Increased HA production by the tumor cells was found to contribute to FGFR-induced proliferation, migration and therapeutic resistance. Finally, analysis of human breast cancer samples reveals a correlation between activated FGFR signaling and tumor-associated expression of HA. In conclusion, our studies demonstrate that FGFR activation leads to STAT3 activity, which regulates expression of Has2 and production of tumor cell-derived HA. Together, these results identify a novel pathway by which FGFR induces proinflammatory signaling pathways and modifies the microenvironment, leading to enhanced FGFR-induced tumor growth. Citation Format: Laura Bohrer, Polly Chuntova, James McCarthy, Kaylee Schwertfeger. STAT3-induced production of hyaluronan contributes to FGFR-induced migration, proliferation, and therapeutic resistance in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B107.