Figure 4 - uploaded by Stephen P Evanko
Content may be subject to copyright.
Hyaluronan cable structure in live cells. (A) Occasional strands in poly I:C-treated cells were visible (arrows) above unfixed cells due to trapped particles of cell debris. (B) The same field as shown in (A). Five minutes after addition of Streptomyces hyaluronidase, the strand is destroyed.
Source publication
We have examined structural details of hyaluronan- and versican-rich pericellular matrices in human lung fibroblasts, as well as fixation effects after treatment with the viral mimetic, poly I:C. Lateral aggregation of hyaluronan chains was promoted by acid-ethanol-formalin fixation compared with a network appearance with formalin alone. However, h...
Citations
... Although HA has previously been implicated in vessel growth stimulation and been applied during bioengineering for vessel tube formation 69,[78][79][80][81][82][83][84] , no studies have reported that HA lines coronary vessels and paves the road for coronary growth during morphogenesis and natural regeneration. These hapln1a + cell-derived HA cables may function as scaffolds, which could prevent loss of ECM components during tissue remodeling, act as a template for matrix regeneration, and support interactions with other cell types [93][94][95][96][97] . Our recent and current studies raise an intriguing possibility that hapln1a + epicardial cells oversee principal cardiogenic activities including myocardial expansion and coronary growth. ...
Although several tissues and chemokines orchestrate coronary formation, the guidance cues for coronary growth remain unclear. Here, we profile the juvenile zebrafish epicardium during coronary vascularization and identify hapln1a⁺ cells enriched with vascular-regulating genes. hapln1a⁺ cells not only envelop vessels but also form linear structures ahead of coronary sprouts. Live-imaging demonstrates that coronary growth occurs along these pre-formed structures, with depletion of hapln1a⁺ cells blocking this growth. hapln1a⁺ cells also pre-lead coronary sprouts during regeneration and hapln1a⁺ cell loss inhibits revascularization. Further, we identify serpine1 expression in hapln1a⁺ cells adjacent to coronary sprouts, and serpine1 inhibition blocks vascularization and revascularization. Moreover, we observe the hapln1a substrate, hyaluronan, forming linear structures along and preceding coronary vessels. Depletion of hapln1a⁺ cells or serpine1 activity inhibition disrupts hyaluronan structure. Our studies reveal that hapln1a⁺ cells and serpine1 are required for coronary production by establishing a microenvironment to facilitate guided coronary growth.
... Lecticans are HA-binding proteoglycans, containing chondroitin sulfate side chains, that ionically bind to HA through clusters of positively charged amino acids forming the link domain (48,53). Little is known about how lecticans are impacted in bacterial pneumonia; however, levels of hyaluronan and the lectican, versican, increase during lung injury (38,80,81), perhaps by HA synthase regulation (82,83). Although rats exposed to fetal alcohol showed a decrease in synaptic versican (84), the role of versican in alcohol-induced lung derangements continue to be an active area of investigation. ...
Although the epidemiology of bacterial pneumonia and excessive alcohol use is well established, the mechanisms by which alcohol induces risk of pneumonia are less clear. Patterns of alcohol misuse, termed alcohol use disorders (AUD), affect about 15 million people in the United States. Compared to otherwise healthy individuals, AUD increase the risk of respiratory infections and acute respiratory distress syndrome (ARDS) by 2-4-fold. Levels and fragmentation of hyaluronic acid (HA), an extracellular glycosaminoglycan of variable molecular weight, are increased in chronic respiratory diseases, including ARDS. HA is largely involved in immune-assisted wound repair and cell migration. Levels of fragmented, low molecular weight HA are increased during inflammation and decrease concomitant with leukocyte levels following injury. In chronic respiratory diseases, levels of fragmented HA and leukocytes remain elevated, inflammation persists, and respiratory infections are not cleared efficiently, suggesting a possible pathological mechanism for prolonged bacterial pneumonia. However, the role of HA in alcohol-induced immune dysfunction is largely unknown. This mini literature review provides insights into understanding the role of HA signaling in host immune defense following excessive alcohol use. Potential therapeutic strategies to mitigate alcohol-induced immune suppression in bacterial pneumonia and HA dysregulation are also discussed.
... With non-activated CD4 T cells, the HA was confined primarily to the cell surface of the FLS and the cells were more elongated (Fig. 1B), whereas in co-cultures with activated CD4 T cells, there was increased staining intensity in the ECM; and the HA was organized into filamentous strands with some cable-like structures throughout the ECM (Fig. 1C). Morphologically, the FLS appeared spread out and flattened, similar to the phenotypes shown in previous studies in which double-stranded RNA-mediated TLR3 activation of fibroblasts increased production of HA [20,21]. In addition, a number of activated CD4 T cells exhibited strong HA staining of their cell surfaces and were associated with the filamentous HA-positive structures (white arrows in Fig. 1C). ...
... However, the impact of the interaction between activated leukocytes such as CD4 T cells and stromal fibroblasts on the generation of a proinflammatory ECM is not yet clear. Recently, we and others have found that stimulation of lung fibroblasts or FLS with inflammatory agonists such as polyinosinicpolycytidylic acid (poly I:C) and respiratory syncytial virus produces elevated levels of HA, which promotes adhesion and activation of CD4 T cells, monocytes, eosinophils, and mast cells [12,20,[48][49][50][51][52][53][54][55][56][57][58][59][60]. Furthermore, we developed a co-culture model of activated CD4 T cells and FLS and found that crosstalk between these two cell types promoted the formation of an HA-rich ECM that was adhesive for leukocytes [19]. ...
... HAPLN3 is a member of the HA-binding link protein family and has widespread expression in various adult tissues [63]. Accumulation of crosslinked forms of HA at sites of inflammation provides a substrate for myeloid and lymphoid cell adhesion by binding to CD44 on the surface of cells [20,[48][49][50][51][52][53][54][55]64]. In other autoimmune diseases such as type 1 diabetes, HA accumulation precedes leukocyte infiltration and is prominent during early insulitis [65]. ...
The content and organization of hyaluronan (HA) in the extracellular matrix (ECM) have been identified as strong indicators of inflammation in joint disease, although the source and role of HA as an effector of inflammation is not clear. In this study, we established co-cultures of activated human CD4 T cells with fibroblast-like synoviocytes (FLS) from osteoarthritis (OA) and rheumatoid arthritis (RA) subjects and examined the role of HA in promoting inflammatory events. Co-cultures of RA FLS with activated CD4 T cells generated an HA-enriched ECM that promoted enhanced monocyte adhesion compared to co-cultures of OA FLS with activated CD4 T cells. In addition, both OA FLS and RA FLS co-cultures with activated CD4 T cells elicited significant increases in the expression of IL1β, TNF, and IL6, with the increase in IL6 expression most prominent in RA co-cultures. Blocking HA synthesis and accumulation with 4-methylumbelliferone reduced expression of IL6, IL1β, and TNF in both OA FLS and RA FLS co-cultures. The increase in HA synthesis in the co-cultures was mimicked by IL6 trans-signaling of FLS in the absence of CD4 T cells. Inhibition of HA synthesis blocked the increase in IL6 by RA FLS mediated by IL6 trans-signaling, suggesting that the HA synthetic pathway may be a key mediator in IL6 expression by FLS. Overall, our study indicates that HA-enriched ECM generated by co-cultures of activated CD4 T cells with FLS from human joints creates a pathogenic microenvironment by promoting adhesion of leukocytes and expression of inflammatory cytokines including IL6.
... A number of imaging techniques have been used for probing HA. For example, histological analysis, through which HA in cell culture or tissues is labeled for direct imaging, has been demonstrated for a wide range of specimens (80)(81)(82)(83)(84)(85)(86). Because HA-specific antibodies are not available (HA is a "self" molecule) (87), labeling of HA has been enabled instead by highly specific HA binding proteins, especially the cartilage link protein (88), the aggrecan terminal fragment globular domain 1-interglobular domain-globular domain 2 (G1-IGD-G2 or HA binding protein, HABP) (89), and VG1 (90). ...
The carbohydrate hyaluronan (or hyaluronic acid, HA) is found in all human tissues and biofluids where it has wide-ranging functions in health and disease that are dictated by both its abundance and size. Consequently, hyaluronan evaluation in physiological samples has significant translational potential. While the analytical tools and techniques for probing other biomolecules like proteins and nucleic acids have become standard approaches in biochemistry, those available for investigating hyaluronan are less well-established. In this review, we survey methods related to the assessment of native hyaluronan in biological specimens, including protocols for separating it from biological matrices and technologies for determining its concentration and molecular weight.
... When FN and collagen are present and strained together, there is a synergistic effect and the energy barrier to collagen fibril assembly is lowered further 41 . The third component molecule, HA, with its very long contour length (~20 um) and thus long molecular relaxation time, is also an excellent candidate for FIA as it does not need very high extensional strain rates to molecularly align with applied strain and it is known to form filaments or "cables" 67 . The most important aspect of MFSC is that structure is formed directly in the path of the forces that create it, ostensibly to resist subsequent forces which propagate along the same direction. ...
During development, mesenchymal cells direct the elaboration of extracellular matrix that shapes the initial animal bauplan which subsequently grows to produce mechanically-competent structure. To gain insight into the processes that initiate matrix formation at the cellular level, high temporal and spatial resolution videos were obtained from a primary human corneal fibroblast (PHCF) cell culture system known to produce an organized, collagenous stroma similar to a human cornea. The images were taken over a 4-day period prior to culture confluency which permitted a clear view of the cell kinematics and any elaborated filaments. The movies reveal an active cellular system in which the PHCFs execute five types of high-velocity and high extensional strain-rate 'pulls' that produce persistent filaments. In four of the pull types, average maximum strain rates (~0.1-0.33s-1) were adequate to induce aggregation and/or crystallization in crowded biopolymer systems. The results demonstrate that PHCFs have the capacity to mechanically induce the formation of biopolymer structures intercellularly and in the path of force.
... The notion that HA configuration is critical for receptor binding is supported by numerous experimental findings. Notably the distinctive pericellular HA cables formed by wrapping of the polymer chains around their binding partner versican and covalent attachment to IαI (inter α trypsin inhibitor) heavy chain present in cells exposed to inflammatory stimuli, convert HA from a weak to a strongly adhesive state for CD44 on monocytes (73,74,75,76). Likewise, in the case of LECs, complexing HA with TSG-6 dramatically enhances its binding to LYVE-1 (65). ...
DCs play a vital role in immunity by conveying antigens from peripheral tissues to draining lymph nodes, through afferent lymphatic vessels. Critical to the process is initial docking to the lymphatic endothelial receptor LYVE-1 via its ligand hyaluronan on the DC surface. How this relatively weak binding polymer is configured for specific adhesion to LYVE-1, however, is unknown. Here, we show that hyaluronan is anchored and spatially organized into a 400–500 nm dense glycocalyx by the leukocyte receptor CD44. Using gene knockout and by modulating CD44-hyaluronan interactions with monoclonal antibodies in vitro and in a mouse model of oxazolone-induced skin inflammation, we demonstrate that CD44 is required for DC adhesion and transmigration across lymphatic endothelium. In addition, we present evidence that CD44 can dynamically control the density of the hyaluronan glycocalyx, regulating the efficiency of DC trafficking to lymph nodes. Our findings define a previously unrecognized role for CD44 in lymphatic trafficking and highlight the importance of the CD44:HA:LYVE-1 axis in its regulation.
... However, production of TSG6 returns to the levels expressed by the PBS controls at 72 h. Does this allow for the space along the HA chains to then be occupied by the more inflammatory chondroitin sulfate versican, which could contribute to continuing inflammation (35,36,(48)(49)(50)? The MO also have the potential themselves to generate low m.w. ...
Early life respiratory syncytial virus (RSV) infection has been linked to the onset of asthma. Despite this association, our knowledge of the progression of the initial viral infection is limited, and no safe or effective vaccine currently exists. Bronchioalveolar lavage, whole-lung cellular isolation, and gene expression analysis were performed on 3-wk- (juvenile) and 8-wk-old (adult) RSV-infected C57BL/6 mice to investigate age-related differences in immunologic responses; juvenile mice displayed a sustained myeloid infiltrate (including monocytes and neutrophils) with increased RNA expression of Ccl2, Ccl3, and Ccl4, when compared with adult mice, at 72 h postinfection. Juvenile mice demonstrated aSma expression (indicative of myofibroblast activity), increased hyaluronan deposition in the lung parenchyma (attributed to asthma progression), and a lack of CD64 upregulation on the surface of monocytes (which, in conjunction with serum amyloid P, is responsible for clearing residual hyaluronan and cellular debris). RSV infection of human airway epithelial cell, human lung fibroblast, and U937 monocyte cocultures (at air-liquid interface) displayed similar CCL expression and suggested matrix metalloproteinase-7 and MMP9 as possible extracellular matrix modifiers. These mouse data, in conjunction with our findings in human monocytes, suggest that the sustained influx of myeloid cells in the lungs of juvenile mice during acute RSV infection could potentiate extracellular matrix remodeling, facilitating conditions that support the development of asthma.
... 19 Binding of Vcan to HA leads to a rich pericellular matrix. 20,21 Haploinsufficient Vcan ∆3/∆3 fibroblasts whose Vcan binds to HA with less affinity attain premature senescence through constitutive activation of EKR1/2 via CD44. 22 Therefore, the action of the G1 domain appears to be mediated by HA and cell surface HA-receptors. ...
Versican is a large chondroitin sulfate/dermatan sulfate proteoglycan belonging to the aggrecan/lectican family. In adults, this proteoglycan serves as a structural macromolecule of the extracellular matrix in the brain and large blood vessels. In contrast, versican is transiently expressed at high levels during development and under pathological conditions when the extracellular matrix dramatically changes, including in the inflammation and repair process. There are many reports showing the upregulation of versican in cancer, which correlates with cancer aggressiveness. Versican has four classical splice variants, and all the variants contain G1 and G3 domains at N- and C-termini, respectively. There are two glycosaminoglycan attachment domains CSα and CSβ. The largest V0 variant contains both CSα and CSβ, V1 contains CSβ, V2 contains CSα, and the shortest G3 variant has neither of them. Versican degradation is initiated by cleavage at a site in the CSβ domain by ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteinases. The N-terminal fragment containing the G1 domain has been reported to exert various biological functions, although its mechanisms of action have not yet been elucidated. In this review, we describe the role of versican in inflammation and cancer and also address the biological function of versikine.
... A potential limitation regarding the present study is caused by the common fixation method with neutralbuffered formalin [11] which may underestimate the HA content of tissue sections [12,13]. Thus, as neutral formaldehyde buffer had been used, the staining observed might show less staining for HA than for samples fixed in other solutions such as ethanol that has been shown to increase HA preservation. ...
Introduction
Tumor necrosis factor-inducible gene 6 protein (TSG-6) is member of the hyaluronan-binding protein family (hyaladherins) to which CD44 also belongs. Inflammatory mediators such as tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1) stimulate TSG-6 production. Recently, however, externally applied TSG-6 has been shown to be effective in the treatment of inflammatory dry eye. On the other hand, it is still unknown whether TSG-6 is naturally present in human corneal epithelium.
Material and methods
Corneal sections of 15 eyes enucleated for posterior segment uveal melanoma were immunohistochemically stained for hyaluronic acid (HA), CD44, and TSG-6.
Results
Throughout the corneal epithelium of all sections, CD44 and hyaluronic acid were detected most intensely in the basal epithelial layer. Whereas the presence of HA was intense even in the cytoplasm of the cells, CD44 was located predominantly at the cell membranes. The intensity of the specific staining decreased towards the surface, where CD44 was barely detectable. Hyaluronic acid was, on the other hand, detectable in the extracellular matrix and cells, even at the surface. TSG-6 like immunoreactivity was detected in all sections in a pattern similar to CD44 but much more distinct and intense, with a marked localization in the cell membranes and intercellular spaces, i.e., extracellular matrix. TSG-6 like immunoreactivity was clearly detectable through all cell layers of the corneal epithelium. All control sections were negative.
Discussion
Tumor necrosis factor-inducible gene 6 (TSG-6)- like protein is present in human corneal epithelium. It might be a natural component of this tissue which is constantly exposed and mechanically traumatized, and displays localization with similarities to that of CD44. The immunohistological detection of HA as major component of the ECM and epithelial tissue only confirms the results of earlier studies. However, the simultaneous presence and colocalization of CD44 and TSG-6, both HA-binding proteins, requires further investigation of the individual role, regulation and interaction of this system.
Conclusion
The detection of TSG-6 in human corneal epithelium in the absence of inflammation underlines the importance of normal mechanical forces on the gene expression and regulation of this protein in ocular surface tissues. Given the relationship between inflammation and the protein, TSG-6 may be a major unknown and underestimated player in the regulation of the inflammation encountered in the presence of ocular surface desiccation and dry eye disease.
... Many previous studies performed by our group and others suggest that these cellular features are connected to each other. Cell types that have especially long and numerous plasma membrane protrusions, such as neuroblastoma cells [30], smooth muscle cells [15], human fibroblasts [31,32], and fibroblasts of Shar Pei dogs with high HAS2 expression [33] have typically high hyaluronan secretion capacity. Even more direct evidence on the effect of hyaluronan synthesis on the growth of filopodia is the rapid regrowth of filopodia after blocking and releasing of hyaluronan synthesis [12,13]. ...
Filopodia are multifunctional finger-like plasma membrane protrusions with bundles of actin filaments that exist in virtually all cell types. It has been known for some time that hyaluronan synthesis activity induces filopodial growth. However, because of technical challenges in the studies of these slender and fragile structures, no quantitative analyses have been performed so far to indicate their association with hyaluronan synthesis. In this work we comprehensively address the direct quantification of filopodial traits, covering for the first time length and density measurements in a series of human cancer cell lines with variable levels of hyaluronan synthesis. The synthesis and plasma membrane binding of hyaluronan were manipulated with hyaluronan synthase 3 (HAS3) and hyaluronan receptor CD44 overexpression, and treatments with mannose, 4-methylumbelliferone (4-MU), and glucosamine. The results of this work show that the growth of filopodia was associated with the levels of hyaluronan synthesis but was not dependent on CD44 expression. The results confirm the hypothesis that abundance and length of filopodia in cancer cells is associated with the activity of hyaluronan synthesis.
