Hyaluronan-rich Extracellular Matrix Research Articles

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration.

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube is an essential process for NCC production and their subsequent migration toward target sites. The migratory route of NCCs, including the surrounding neural tube tissues, involves hyaluronan (HA)-rich extracellular matrix. To model NCC migration into these HA-rich surrounding tissues from the neural tube, a mixed substrate migration assay consisting of HA (average molecular weight: 1,200-1,400 kDa) and collagen type I (Col1) was established in this study. This migration assay demonstrates that NCC cell line, O9-1, cells are highly migratory on the mixed substrate and that the HA coating is degraded at the site of focal adhesions in the course of migration. This in vitro model can be useful for further exploration of the mechanistic basis involved in NCC migration. This protocol is also applicable for evaluating different substrates as scaffolds to study NCC migration.

4-Methylumbelliferone Decreases the Hyaluronan-rich Extracellular Matrix and Increases the Effectiveness of 5-Fluorouracil.

Pancreatic cancer responds poorly to most chemotherapeutic agents. Several studies have reported that hyaluronan (HA)-rich extracellular matrix (ECM) is a biological barrier against chemotherapeutic agents. 4-methylumbelliforone (MU) led to inhibition of HA synthesis and its preservation in ECM, which may enhance 5-fluorouracil (5-FU) cytotoxicity. Thus, new therapy with MU and 5-FU may be developed for pancreatic cancer. A 5-fluorouracil (5-FU) concentration and 4-methylumbelliferone (MU) dosage was analyzed by high-performance liquid chromatography (HPLC). Change in antitumor efficacy of 5-FU in combination with MU was also examined in vivo and in vitro. Combined 5-FU and MU treatment inhibited cell proliferation better than 5-FU alone; 0.01 mM 5-FU alone decreased cell proliferation by 37.7 %, while 0.01 mM 5-FU with 0.5 mM MU decreased cell proliferation by 57.4%. MU enhanced the intracellular concentration of 5-FU by 47.3% compared to control. Mice tumors treated with 5-FU and MU decreased in size and animal survival was prolonged. Moreover, MU decreased cohesiveness of the intercellular space. Combination therapy of 5-FU with MU was effective. A novel therapy can be designed for pancreatic cancer by using ECM modulation.

The fasciacytes: A new cell devoted to fascial gliding regulation.

Hyaluronan occurs between deep fascia and muscle, facilitating gliding between these two structures, and also within the loose connective tissue of the fascia, guaranteeing the smooth sliding of adjacent fibrous fascial layers. It also promotes the functions of the deep fascia. In this study a new class of cells in fasciae is identified, which we have termed fasciacytes, devoted to producing the hyaluronan-rich extracellular matrix. Synthesis of the hyaluronan-rich matrix by these new cells was demonstrated by Alcian Blue staining, anti-HABP (hyaluronic acid binding protein) immunohistochemistry, and transmission electron microscopy. Expression of HAS2 (hyaluronan synthase 2) mRNA by these cells was detected and quantified using real time RT-PCR. This new cell type has some features similar to fibroblasts: they are positive for the fibroblast marker vimentin and negative for CD68, a marker for the monocyte-macrophage lineage. However, they have morphological features distinct from classical fibroblasts and they express the marker for chondroid metaplasia, S-100A4. The authors suggest that these cells represent a new cell type devoted to the production of hyaluronan. Since hyaluronan is essential for fascial gliding, regulation of these cells could affect the functions of fasciae so they could be implicated in myofascial pain. Clin. Anat. 31:667-676, 2018. © 2018 Wiley Periodicals, Inc.

Adult human retinal Müller glia display distinct peripheral and macular expression of CD117 and CD44 stem cell-associated proteins

Experimental evidence suggests human Müller glia exhibit neural progenitor properties in vitro. CD117 and CD44 are known to be expressed by stem cells, the survival of which appears to depend critically on interactions with hyaluronan-rich extracellular matrix (ECM). Here, we characterise Müller glia expression of CD117 and CD44 in normal adult human retina and describe how it correlates with hyaluronan distribution in ocular ECM. By using chromogen-based immunohistochemistry, CD117 expression was found in entire Müller glia cytoplasm spanning from inner to outer limiting membrane in both peripheral retina (PR) and macular retina (MR), mirroring expression of the established Müller glia marker vimentin. Unlike vimentin, CD117 was also strongly expressed by Müller glia nuclei. Relative to total inner nuclear layer (INL) nuclei, more CD117+ Müller glia nuclei were seen in PR than MR. By contrast, CD44 expression was found predominantly in Müller glia apical processes of PR; no expression was found in MR. Astral blue staining demonstrated the presence of hyaluronan in cortical vitreous and the interphotoreceptor matrix (IPM) in both MR and PR. Our findings demonstrate that: (i) both CD117 and CD44 are expressed by human adult Müller glia; (ii) CD117 is a robust nuclear and cytoplasmic immunohistochemical marker of Müller glia; and (iii) that while CD117 is expressed by the entire Müller glia in both PR and MR, CD44 is only expressed by Müller glia apices in PR. Since the apices of Müller glia are in direct contact with the hyaluronan-rich IPM, the Müller glia-IPM interface in PR is likely a favourable region for supporting progenitor or stem cell-like signalling. These observations provide novel insights into potential stem-cell favouring microenvironments in mature human retina.

Cyclic AMP-elevating Agents Promote Cumulus Cell Survival and Hyaluronan Matrix Stability, Thereby Prolonging the Time of Mouse Oocyte Fertilizability

Cumulus cells sustain the development and fertilization of the mammalian oocyte. These cells are retained around the oocyte by a hyaluronan-rich extracellular matrix synthesized before ovulation, a process called cumulus cell-oocyte complex (COC) expansion. Hyaluronan release and dispersion of the cumulus cells progressively occur after ovulation, paralleling the decline of oocyte fertilization. We show here that, in mice, postovulatory changes of matrix are temporally correlated to cumulus cell death. Cumulus cell apoptosis and matrix disassembly also occurred in ovulated COCs cultured in vitro. COCs expanded in vitro with FSH or EGF underwent the same changes, whereas those expanded with 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP) maintained integrity for a longer time. It is noteworthy that 8-Br-cAMP treatment was also effective on ovulated COCs cultured in vitro, prolonging the vitality of the cumulus cells and the stability of the matrix from a few hours to >2 days. Stimulation of endogenous adenylate cyclase with forskolin or inhibition of phosphodiesterase with rolipram produced similar effects. The treatment with selective cAMP analogues suggests that the effects of cAMP elevation are exerted through an EPAC-independent, PKA type II-dependent signaling pathway, probably acting at the post-transcriptional level. Finally, overnight culture of ovulated COCs with 8-Br-cAMP significantly counteracted the decrease of fertilization rate, doubling the number of fertilized oocytes compared with control conditions. In conclusion, these studies suggest that cAMP-elevating agents prevent cumulus cell senescence and allow them to continue to exert beneficial effects on oocyte and sperm, thereby extending in vitro the time frame of oocyte fertilizability.

Abstract 4999: Hyaluronan-rich ECM contributes to resistance to antibody-dependent cell-mediated cytotoxicity in solid tumors.

Abstract Accumulation of Hyaluronan (HA), an important glycosaminoglycan of the extracellular matrix (ECM), has been detected in about 25% of human tumors, and 56% of breast cancers1-2. HA is produced by malignant and stromal cells and acts to cross-link ECM proteins, contributing to the desmoplastic phenotype. Aberrant accumulation of HA in tumors has been associated with more aggressive malignancy1-2. We have observed that >50% of human epidermal growth factor receptor 2 (HER2) triple-positive breast tumors have a high accumulation of HA (HA3+). Monoclonal antibodies (MAbs) have been developed successfully as part of the anticancer armamentarium. However, even in cases where the target of the antibody is stably and abundantly expressed, efficacy often falls below expectations. In this work, we present a novel HA-dependent ECM-mediated mechanism of resistance to antibody-dependent cell-mediated cytotoxicity (ADCC) and this resistance can be reduced by PEGPH20 (a pegylated human PH20) treatment. Human hyaluronan synthase 2 (HAS2)-overexpressing tumor cells formed an HA-rich pericellular matrix which can restrict human NK cells from accessing tumor cells. This HA-dependent ECM-mediated physical barrier in HAS2- overexpressing tumor cells contributed to resistance to trastuzumab/cetuximab-dependent ADCC. Removal of HA from ECM by PEGPH20 treatment sensitized HAS2-overexpressing tumor cells to trastuzumab/cetuximab-mediated ADCC in vitro. HAS2-overexpressing ovarian cancer cell SKOV3 showed more aggressive growth pattern (median survival on 42 days) compared to parental SKOV3 (median survival of 61 days) in an intra-peritoneal xenograft model. In the same model with HAS2-overexpressing SKOV3, PEGPH20 in combination with trastuzumab and NK cell treatment (to simulate ADCC in vivo) resulted in 70% tumor growth inhibition (TGI) in comparison to 40% TGI with trastuzumab and NK cell treatment, indicating that PEGPH20 enhanced in vivo ADCC by trastuzumab and NK cell. Our results demonstrated a mechanism of high HA-content pericellular matrix-mediated resistance to MAb-mediated ADCC. These results may help explain why tumors with high levels of HA (HA3+ phenotype) are more aggressive, and suggest that removal of HA by PEGPH20 treatment may be an effective combinatorial therapy together with anti-cancer MAbs. 1. Kultti A, Li X, Jiang P, Thompson C.B., Frost G.I., Shepard H.M. 2012. Therapeutic targeting of Hyaluronan in the tumor stroma. Cancers, 4:873-903; doi:10.3390/cancers4030873. 2. Sironen, R.K., Tammi, M., Tammi, R., Auvinen, P.K., Anttila, M., and Kosma, V.M. 2011. Hyaluronan in human malignancies. Exp. Cell. Res. 317:383-391. doi: 10.1016/j.yexcr.2010.11.017 Citation Format: Netai C. Singha, Tara Nekoroski, Susan Zimmerman, Chunmei Zhao, Ping Jiang, Robert Connor, Gregory I. Frost, Zhondong Huang, Michael H. Shepard. Hyaluronan-rich ECM contributes to resistance to antibody-dependent cell-mediated cytotoxicity in solid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4999. doi:10.1158/1538-7445.AM2013-4999

Role of interleukins in the regulation of ovarian functions

Reproduction is a result of coordinated signaling network between gonads and pituitary/hypothalamus. Ovarian functions, such as follicular development, ovulation, luteinisation, luteolysis, and remodeling of the endometrium, are controlled by endocrine, paracrine and autocrine factors. Ovulation is a unique biological process by which the complex of a mature oocyte and surrounding somatic cumulus cells (CCs), named oocyte-cumulus complex (OCC), is released from the follicle into the oviduct for transport and fertilization. Recently, evidence has been accumulated that the immune system might represent an additional local regulator of the ovarian functions that are essentially modulated by gonadotropins. Moreover, the ovulation is similar to an inflammatory response: follicles become hyperemic, produce prostaglandins (PGs), and synthesize a hyaluronan-rich extracellular matrix. Cytokines are originally referred as numerous signaling substances secreted by certain cells of the immune system which influence the activity of other cells. A number of studies have shown that cytokines may modulate ovarian functions and play an important role in the ovulation. The most known cytokines related to the reproduction are interleukins (ILs). These molecules have been localized in the reproduction-related body fluids and various ovarian cell types, such as the oocytes, granulosa (GCs) and theca cells (TCs) in several mammalian species. Moreover, macrophages in the ovary have been shown to secrete cytokines, including ILs. The present review summarizes the current knowledge on ILs in regard of their role in the regulation of selected ovarian functions. cytokines, interleukins, ovary, steroidogenesis, ovulation, corpus luteum.

Pentraxins and Atherosclerosis: The Role of PTX3

Pentraxins are a family of evolutionarily conserved multifunctional pattern-recognition proteins characterized by a cyclic multimeric structure. Based on the primary structure of the subunit, the pentraxins are divided into two groups: short pentraxins and long pentraxins. C-reactive protein (CRP) and serum amyloid P-component (SAP) are the two short pentraxins. The prototype protein of the long pentraxin group is pentraxin 3 (PTX3). CRP and SAP are produced primarily in the liver in response to IL-6, while PTX3 is produced by a variety of tissues and cells and in particular by innate immunity cells in response to proinflammatory signals and Toll-like receptor (TLR) engagement. PTX3 interacts with several ligands, including growth factors, extracellular matrix components and selected pathogens, playing a role in complement activation and facilitating pathogen recognition by phagocytes, acting as a predecessor of antibodies. In addition, PTX3 is essential in female fertility by acting as a nodal point for the assembly of the cumulus oophorus hyaluronan-rich extracellular matrix. Here we will concisely review the general properties of PTX3 in the context of the pentraxin superfamily and discuss recent data suggesting that PTX3 plays a cardiovascular protective effect. PTX3 may represent a new marker in vascular pathology which correlates with the risk of developing vascular events.

256 ROLE OFTHE BOVINE OOCYTE-SPECIFIC PROTEIN JY-1 IN REGULATION OF MESSENGER RNA ABUNDANCE FOR GENES LINKED TO CUMULUS EXPANSION

We have previously demonstrated a requirement of the oocyte-specific protein JY-1 for oocyte and early embryonic development in cattle. Microin- jection of JY-1 siRNA into cumulus-enclosed germinal vesicle-stage oocytes impedes progression to metaphase II and cumulus expansion during in vitro maturation and limits subsequent embryonic development following in vitro fertilization. Negative effects of siRNA-mediated reduction in JY-1 on oocyte maturation, cumulus expansion, and initial cleavage divisions following in vitro fertilization can be rescued bysupplementation with recom- binant JY-1 protein during oocyte culture. However, the mechanisms involved in JY-1 regulation of above developmental endpoints are unknown. The objective of the current study was to determine whether JY-1-induced regulation of cumulus expansion during meiotic maturation is linked to alterations in mRNA abundance for genes that promote formation and stabilization of the mucified extracellular matrix characteristic of an expanded cumulus layer. Cumulus-enclosed germinal vesicle stage-bovine oocytes were microinjected with JY-1 siRNA, subjected to negative control siRNA microinjection or served as uninjected controls (n = 10 oocytes/treatment; n = 5 replicates), and cultured for 48 h in maturation medium containing 50 μM S-roscovitine to block spontaneous germinal vesicle breakdown. Cumulus-oocyte complexes were then washed and in vitro matured for an additional 24 h in maturation medium minus S-roscovitine. Additional JY-1 siRNA injected and uninjected cumulus-enclosed oocytes were cultured as described earlier but in the presence of 1 ng/mL of recombinant JY-1 protein (n = 10 oocytes/treatment; n = 5 replicates). Dose of recombinant JY-1 protein utilized was previously shown to reverse inhibitory effects of JY-1 siRNA injection on cumulus expansion. After in vitro maturation, cumulus cells were harvested and RNA isolated and subjected to reverse transcription. Real-time PCR analysis was then conducted to determine the effect of treatments on cumulus-cell mRNA abundance for HAS2, HAS3, PTX3, and TNFAIP6. Effects of JY-1 supplementation or depletion (siRNA injection) on cumulus-cell mRNA abundance for HAS2 and HAS3 (enzymes involved in hyaluronan synthesis) were not observed. However, abundance of mRNA for TNFAIP6 and PTX3 (molecules implicated in stabilization of the hyaluronan-rich extracellular matrix) was reduced (relative to uninjected and negative control siRNA groups) in response to JY-1 siRNA injection (P < 0.05) and effects of JY-1 siRNA were rescued by JY-1 protein treatment (P < 0.05). Effects of JY-1 protein supplementation on cumulus-cell TNFAIP6 and PTX3 mRNA in uninjected controls were not observed. Results support a requirement of the oocyte-specific protein JY-1 for regulation of expression of genes functionally linked to stabilization of the hyaluronan-rich extracellular matrix and cumulus expansion. This research was supported by USDA 2008-35203-19094 to G. W. Smith.

037. IMMUNE-LIKE MECHANISMS ASSOCIATED WITH OVULATION

Ovulation is the unique biological process by which a mature oocyte and surrounding somatic cells, the cumulus cell-oocyte complex (COC), are released from the surface of the ovary into the oviduct for transport and fertilization. Ovulation is similar to an inflammatory response: the follicles become hyperemic, produce prostaglandins and synthesize a hyaluronan-rich extracellular matrix. However, this view of ovulation may be too restrictive and need to be broadened to encompass the innate immune cell surveillance response system. This hypothesis is being proposed because ovarian granulosa cells and cumulus cells express and respond to innate immune cell related surveillance proteins (Toll-like receptors 2 and 4) and cytokines such as interleukin 6 (IL6) during ovulation. In addition, recent studies indicate that the ovulation process that is set in motion by the surge of luteinizing hormone (LH) is mediated, in large part, by the EGF-like factors (Amphiregulin, epiregulin and betacellulin) and their critical activation of RAS, most probably KRAS that is expressed at high levels in granulosa cells, and the mitogen activated protein kinases, MAP3/1 (ERK1/2). Mice in which granulosa cells are depleted of ERK1/2 fail to ovulate, oocyte meiosis does not resume, COC expansion is impaired and luteinization is blocked. Thus the global molecular reprogramming of granulosa cell gene expression patterns is completely derailed. Supported, in part by NIH-HD-16229, -16272 and -07495 (SCCPIR).

Immune-like mechanisms in ovulation

Ovulation is the unique biological process by which a mature oocyte (egg) and surrounding somatic cells, the cumulus cell-oocyte complex (COC), are released from the surface of the ovary into the oviduct for transport and fertilization. Ovulation is similar to an inflammatory response: the follicles become hyperemic, produce prostaglandins and synthesize a hyaluronan-rich extracellular matrix. However, this view of ovulation might be too restrictive and needs to be broadened to encompass the innate immune cell surveillance-response system. This hypothesis is being proposed because ovarian granulosa cells and cumulus cells express and respond to innate immune cell-related surveillance proteins (Toll-like receptors 2 and 4) and cytokines, such as interleukin 6 (IL-6), during ovulation.

PKC-induced stiffening of hyaluronan/CD44 linkage; local force measurements on glioma cells

Interaction of cells with hyaluronan (HA) rich extracellular matrix involves the membrane receptor CD44. HA–CD44 interactions are particularly important in the development of glioma pathogenesis for its implication in tumor cells spreading. Highly motile states rely on the spaciotemporal regulation of HA–CD44 interactions occurring in specific cytoskeletal-supported membrane organization such as microvilli or the leading edge observed in migrating cell. We used AFM-based force measurement to probe the HA–CD44 interaction at localized regions at the surface of living glioma cells expressing high level of the CD44 standard isoform. We show that unstimulated cells interact with HA over their entire surfaces and are highly deformable when force is exerted on individual HA molecules bound to membrane CD44 receptors. Conversely, in PKC-activated cells the probed interactions are concentrated at the leading edge of the cells with reduced membrane deformability. Taken together, our results show that PKC-enhanced motility in glioma cells is associated with a redistribution of CD44 receptors at the leading edges concomitant with a stiffer anchoring of CD44 to the cell surface involving the actin cytoskeleton.

The Long Pentraxin PTX3, a Soluble Pattern Recognition Receptor Involved in Innate Immunity,Inflammation and Female Fertility

Pentraxins are a family of evolutionarily conserved multifunctional pattern-recognition proteins characterized by a cyclic multimeric structure. Based on the primary structure of the subunit, the pentraxins are divided into two groups: short pentraxins and long pentraxins. C-reactive protein (CRP) and serum amyloid P-component (SAP) are the two short pentraxins. The prototype protein of the long pentraxin group is pentraxin 3 (PTX3). CRP and SAP are produced primarily in the liver in response to IL-6, while PTX3 is produced by a variety of tissues and cells and in particular, by innate immunity cells in response to proinflammatory signals and Toll-like receptor (TLR) engagement. PTX3 interacts with several ligands, including growth factors, extracellular matrix components and selected pathogens, playing a role in complement activation and facilitating pathogen recognition by phagocytes, acting as a predecessor of antibodies. In addition, PTX3 is essential in female fertility by acting as a nodal point for the assembly of the cumulus oophorus hyaluronan-rich extracellular matrix. Thus, the prototypic long pentraxin PTX3 is a multifunctional soluble pattern recognition receptor acting as a nonredundant component of the humoral arm of innate immunity and involved in tuning inflammation, in matrix deposition and female fertility. Keywords: Pentraxins, innate immunity, inflammation, marker

The long pentraxin PTX3 in vascular pathology

Pentraxins are a family of evolutionarily conserved multifunctional pattern-recognition proteins characterized by a cyclic multimeric structure. Based on the primary structure of the subunit, the pentraxins are divided into two groups: short pentraxins and long pentraxins. C-reactive protein (CRP) and serum amyloid P-component (SAP) are the two short pentraxins. The prototype protein of the long pentraxin group is pentraxin 3 (PTX3). CRP and SAP are produced primarily in the liver in response to IL-6, while PTX3 is produced by a variety of tissues and cells and in particular by innate immunity cells in response to proinflammatory signals and Toll-like receptor (TLR) engagement. PTX3 interacts with several ligands, including growth factors, extracellular matrix components and selected pathogens, playing a role in complement activation and facilitating pathogen recognition by phagocytes, acting as a predecessor of antibodies. In addition, PTX3 is essential in female fertility by acting as a nodal point for the assembly of the cumulus oophorus hyaluronan-rich extracellular matrix. Thus, the prototypic long pentraxin PTX3 is a multifunctional soluble pattern recognition receptor acting as a non-redundant component of the humoral arm of innate immunity and involved in tuning inflammation, in matrix deposition and female fertility.

The long pentraxin PTX3 as a link among innate immunity, inflammation, and female fertility

The long pentraxin 3 (PTX3) is member of a complex superfamily of multifunctional proteins characterized by a cyclic multimeric structure. PTX3 is highly conserved in evolution and is produced by innate-immunity cells in response to proinflammatory signals and Toll-like receptor engagement. PTX3 plays complex, nonredundant functions in vivo, acting as a predecessor of antibodies, recognizing microbes, activating complement, facilitating pathogen recognition by phagocytes, and hence, playing a nonredundant role in resistance against selected pathogens. In addition, PTX3 is essential in female fertility by acting as a nodal point for the assembly of the cumulus oophorus hyaluronan-rich extracellular matrix. Thus, the prototypic long pentraxin PTX3 is a multifunctional, soluble pattern recognition receptor acting as a nonredundant component of the humoral arm of innate immunity and involved in matrix deposition and female fertility.

PENTRAXINS AT THE CROSSROADS BETWEEN INNATE IMMUNITY, INFLAMMATION, MATRIX DEPOSITION, AND FEMALE FERTILITY

C reactive protein, the first innate immunity receptor identified, and serum amyloid P component are classic short pentraxins produced in the liver. Long pentraxins, including the prototype PTX3, are expressed in a variety of tissues. Some long pentraxins are expressed in the brain and some are involved in neuronal plasticity and degeneration. PTX3 is produced by a variety of cells and tissues, most notably dendritic cells and macrophages, in response to Toll-like receptor (TLR) engagement and inflammatory cytokines. PTX3 acts as a functional ancestor of antibodies, recognizing microbes, activating complement, and facilitating pathogen recognition by phagocytes, hence playing a nonredundant role in resistance against selected pathogens. In addition, PTX3 is essential in female fertility because it acts as a nodal point for the assembly of the cumulus oophorus hyaluronan-rich extracellular matrix. Thus, the prototypic long pentraxin PTX3 is a multifunctional soluble pattern recognition receptor at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility.

Enhanced neutrophil extravasation and rapid progression of proteoglycan-induced arthritis in TSG-6-knockout mice.

To gain insight into the mechanisms of the antiinflammatory effect of tumor necrosis factor alpha (TNFalpha)-induced protein 6 (Tnfip6) in arthritis, using Tnfip6-deficient animals. TNFalpha-stimulated gene 6 (TSG-6) coding for Tnfip6 was disrupted. Tnfip6-deficient mice were backcrossed into proteoglycan-induced arthritis (PGIA)-susceptible BALB/c mice, and arthritis was induced by systemic immunization with cartilage proteoglycan (PG). Thioglycollate-induced sterile peritonitis was also assessed, to monitor the early events of neutrophil extravasation in wild-type and Tnfip6-deficient mice in the presence or absence of treatment with recombinant murine Tnfip6. The onset of PGIA was similar, but progression and severity were significantly greater, in Tnfip6-deficient mice compared with wild-type BALB/c mice. However, this was not associated with enhanced T or B cell responses to cartilage PGs, but rather, an early and more extensive infiltration of the synovium with neutrophil leukocytes was the most prominent histopathologic feature of PGIA in Tnfip6-deficient mice. This was accompanied by elevated serum levels of interleukin-6 and amyloid A, and significantly increased activities of the enzymes plasmin, myeloperoxidase, and neutrophil elastase in the inflamed paw joints of Tnfip6-null mice, when compared with that of the wild-type littermates. Loss of control over several components of inflammation resulted in extensive and rapid cartilage degradation, bone erosion, joint ankylosis, and deformities in Tnfip6-null animals. In support of the antiinflammatory effect of Tnfip6 via the inhibition of polymorphonuclear (PMN) cell efflux, neutrophil invasion during thioglycollate-induced peritonitis was 2-fold higher in Tnfip6-deficient animals than in wild-type animals, but was dramatically suppressed by intravenous injection of recombinant murine Tnfip6. Tnfip6 is a multifunctional antiinflammatory protein that is produced at the site of inflammation and can be retained by the hyaluronan-rich extracellular matrix. A major effect of Tnfip6 is the inhibition of the extravasation of PMN cells, predominantly neutrophils, into the site of inflammation, most likely via a CD44/hyaluronan/Tnfip6-mediated blocking mechanism.

Endoplasmic Reticulum Stress Induces Hyaluronan Deposition and Leukocyte Adhesion

There is mounting evidence that perturbations in endoplasmic reticulum (ER) function play a key role in the pathogenesis of a broad range of diseases. We have examined the ability of ER stress to modulate leukocyte binding to colonic and aortic smooth muscle cells. In vitro, control smooth muscle cells bind few leukocytes, but treatment with compounds that induce ER stress, including tunicamycin, A23187, and thapsigargin, promotes leukocyte binding. Likewise, dextran sulfate, another agent capable of inducing ER stress and promoting inflammation in vivo, strongly induces leukocyte adhesion. The bound leukocytes are released by hyaluronidase treatment, indicating a critical role for hyaluronan-containing structures in mediating leukocyte binding. Affinity histochemistry demonstrated that hyaluronan accumulates and is present in cable-like structures in the treated, but not the untreated, cultures and that these structures serve as attachment sites for leukocytes. Hyaluronan-rich regions of both murine and human inflamed colon contain numerous cells that stain intensely for ER-resident chaperones containing the KDEL sequence, demonstrating a relationship between ER stress and hyaluronan deposition in vivo. These results indicate that ER stress may contribute to chronic inflammation by forming a hyaluronan-rich extracellular matrix that is conducive to leukocyte binding.

Perivitelline space: does it play a role in blocking polyspermy in mammals?

The perivitelline space of mammalian oocytes changes in size and composition during preimplantation development. Often overlooked in the past, this space contains a hyaluronan-rich extracellular matrix prior to fertilization and a cortical granule envelope following release of the cortical granules at fertilization. The hyaluronan-containing matrix of unfertilized oocytes is well developed in some species such as opossums and humans but is scant in rodents including the hamster and mouse. The significance of the hyaluronan-rich matrix, which attaches to the plasma membrane of the oocytes, is not fully understood. However, hyaluronan, which can inhibit membrane fusion, is present in the perivitelline space (PVS) of unfertilized oocytes and must be negotiated by the fertilizing sperm. Following fertilization, the cortical granule envelope forms as the cortical granules disperse, thereby causing the PVS to increase significantly in size. Calcium is important in the dispersion of the cortical granules following exocytosis. Once formed, the cortical granule envelope in some species is about the same thickness as the zona pellucida, but it is not readily visualized unless it is stained with fluorescent probes or examined ultrastructurally after following stabilization with ruthenium red. The envelope contains proteins that remain in the PVS until the time of blastocyst hatching. Although little work has been done on the functions of the cortical granule envelope, several studies are consistent with the idea that it plays a role in blocking polyspermy. While nicotine increases polyspermy in sea urchins, its effects on polyspermy in human smokers have not been characterized, but could be addressed in human in vitro fertilization labs.

Impaired cumulus mucification and female sterility in tumor necrosis factor-induced protein-6 deficient mice.

Mucification of the cumulus layer around the oocyte is an obligatory process for female fertility. Tumor necrosis factor-induced protein-6 (TNFIP6 or TSG6) has been shown to be specifically expressed during this process. We have generated TNFIP6-deficient mice and tested the ability of their cumulus cells to undergo mucification. Cumulus cell-oocyte complexes fail to expand in TNFIP6-deficient female mice because of the inability of the cumulus cells to assemble their hyaluronan-rich extracellular matrix. The impaired cumulus matrix formation is due to the lack of covalent complexes between hyaluronan and the heavy chains of the inter-alpha-trypsin inhibitor family. As a consequence, TNFIP6-deficient females are sterile. Cultured TNFIP6-deficient cumulus cell-oocyte complexes also fail to expand when stimulated with dibutyryl cyclic AMP or epidermal growth factor. Recombinant TNFIP6 is able to catalyze the covalent transfer of heavy chains to hyaluronan in a cell-free system, restore the expansion of Tnfip6-null cumulus cell-oocyte complexes in vitro, and rescue the fertility in Tnfip6-null females. These results provide clear evidence that TNFIP6 is a key catalyst in the formation of the cumulus extracellular matrix and indispensable for female fertility.