Monocyte adhesive hyaluronan matrix induced by hyperglycemia in diabetic lung injuries.

Infiltrated pre-inflammatory monocytes and macrophages have important roles in the induction of diabetic lung injuries, but the mechanism mediating their infiltration is still unclear. Here, we showed that airway smooth muscle cells (SMCs) activated monocyte adhesion in response to hyperglycemic glucose (25.6 mM) by significantly increasing hyaluronan (HA) in the cell matrix, with concurrent 2- to 4-fold increases in adhesion of U937 monocytic-leukemic cells. The HA-based structures were attributed directly to the high-glucose and not to increased extracellular osmolality, and they required growth stimulation of SMCs by serum. Treatment of SMCs with heparin in high-glucose induces synthesis of a much larger HA matrix, consistent with our observations in the glomerular SMCs. Further, we observed increases in tumor necrosis factor-stimulated gene-6 (TSG-6) expression in high-glucose and high-glucose plus heparin cultures, and the heavy chain (HC)-modified HA structures existed on the monocyte-adhesive cable structures in high-glucose and in high-glucose plus heparin-treated SMC cultures. Interestingly, these HC-modified HA structures were unevenly distributed along the HA cables. Further, the in vitro assay with recombinant human TSG-6 and the HA14 oligo showed that heparin has no inhibitory activity on the TSG-6-induced HC-transfer to HA, consistent with the results from SMC cultures. These results support the hypothesis that hyperglycemia in airway smooth muscle induces the synthesis of a HA matrix that recruits inflammatory cells and establishes a chronic inflammatory process and fibrosis that lead to diabetic lung injuries.

Aggrecan and Hyaluronan: The Infamous Cartilage Polyelectrolytes - Then and Now.

Cartilages are unique in the family of connective tissues in that they contain a high concentration of the glycosaminoglycans, chondroitin sulfate and keratan sulfate attached to the core protein of the proteoglycan, aggrecan. Multiple aggrecan molecules are organized in the extracellular matrix via a domain-specific molecular interaction with hyaluronan and a link protein, and these high molecular weight aggregates are immobilized within the collagen and glycoprotein network. The high negative charge density of glycosaminoglycans provides hydrophilicity, high osmotic swelling pressure and conformational flexibility, which together function to absorb fluctuations in biomechanical stresses on cartilage during movement of an articular joint. We have summarized information on the history and current knowledge obtained by biochemical and genetic approaches, on cell-mediated regulation of aggrecan metabolism and its role in skeletal development, growth as well as during the development of joint disease. In addition, we describe the pathways for hyaluronan metabolism, with particular focus on the role as a "metabolic rheostat" during chondrocyte responses in cartilage remodeling in growth and disease.Future advances in effective therapeutic targeting of cartilage loss during osteoarthritic diseases of the joint as an organ as well as in cartilage tissue engineering would benefit from 'big data' approaches and bioinformatics, to uncover novel feed-forward and feed-back mechanisms for regulating transcription and translation of genes and their integration into cell-specific pathways.

Heparin and calreticulin interact on the surface of early G0/G1 dividing rat mesangial cells to regulate hyperglycemic glucose-induced responses.

Heparin can block pathological responses associated with diabetic nephropathy in animal models and human patients. Our previous studies showed that the interaction of heparin on the surface of rat mesangial cells (RMCs) entering G1 of cell division in hyperglycemic glucose: 1) blocked glucose uptake by glucose transporter 4; 2) inhibited cytosolic uridine diphosphate-glucose elevation that would occur within 6 h from G0/G1; and 3) prevented subsequent activation of hyaluronan synthesis in intracellular compartments and subsequent inflammatory responses. However, specific proteins that interact with heparin are unresolved. Here, we showed by live cell imaging that fluorescent heparin was rapidly internalized into the cytoplasm and then into the endoplasmic reticulum, Golgi, and nuclei compartments. Biotinylated-heparin was applied onto the surface of growth arrested G0/G1 RMCs in order to extract heparin-binding protein(s). SDS-PAGE gels showed two bands at ∼70 kDa in the extract that were absent when unlabeled heparin was used to compete. Trypsin digests of the bands were analyzed by MS and identified as calreticulin and prelamin A/C. Immunostaining with their antibodies identified the presence of calreticulin on the G0/G1 RMC cell surface. Previous studies have shown that calreticulin can be on the cell surface and can interact with the LDL receptor-related protein, which has been implicated in glucose transport by interaction with glucose transporter 4. Thus, cell surface calreticulin can act as a heparin receptor through a mechanism involving LRP1, which prevents the intracellular responses in high glucose and reprograms the cells to synthesize an extracellular hyaluronan matrix after division.

Chemotherapy induces feedback up-regulation of CD44v6 in colorectal cancer initiating cells through ß-catenin/MDR1 signaling to sustain chemoresistance.

Chemoresistance in colorectal cancer initiating cells (CICs) involves the sustained activation of multiple drug resistance (MDR) and WNT/ß-catenin signaling pathways, as well as of alternatively spliced-isoforms of CD44 containing variable exon-6 (CD44v6). In spite of its importance, mechanisms underlying the sustained activity of WNT/ß-catenin signaling have remained elusive. The presence of binding elements of the ß-catenin-interacting transcription factor TCF4 in the MDR1 and CD44 promoters suggests that crosstalk between WNT/ß-catenin/TCF4-activation and the expression of the CD44v6 isoform mediated by FOLFOX, a first-line chemotherapeutic agent for colorectal cancer, could be a fundamental mechanism of FOLFOX resistance. Our results identify that FOLFOX treatment induced WNT3A secretion, which stimulated a positive feedback loop coupling ß-catenin signaling and CD44v6 splicing. In conjunction with FOLFOX induced WNT3A signal, specific CD44v6 variants produced by alternative splicing subsequently enhance the late wave of WNT/ß-catenin activation to facilitate cell cycle progression. Moreover, we revealed that FOLFOX-mediated sustained WNT signal requires the formation of a CD44v6-LRP6-signalosome in caveolin microdomains, which leads to increased FOLFOX efflux. FOLFOX-resistance in colorectal CICs occurs in the absence of tumor-suppressor disabled-2 (DAB2), an inhibitor of WNT/ß-catenin signaling. Conversely, in sensitive cells, DAB2 inhibition of WNT-signaling requires interaction with a clathrin containing CD44v6-LRP6-signalosome. Furthermore, full-length CD44v6, once internalized through the caveolin-signalosome, is translocated to the nucleus where in complex with TCF4, it binds to ß-catenin/TCF4-regulated MDR1, or to CD44 promoters, which leads to FOLFOX-resistance and CD44v6 transcription through transcriptional-reprogramming. These findings provide evidence that targeting CD44v6-mediated LRP6/ß-catenin-signaling and drug efflux may represent a novel approach to overcome FOLFOX resistance and inhibit tumor progression in colorectal CICs. Thus, sustained drug resistance in colorectal CICs is mediated by overexpression of CD44v6, which is both a functional biomarker and a therapeutic target in colorectal cancer.

Transforming Growth Factor-ß Receptor-Mediated, p38 Mitogen-Activated Protein Kinase-Dependent Signaling Drives Enhanced Myofibroblast Differentiation during Skin Wound Healing in Mice Lacking Hyaluronan Synthases 1 and 3.

Normal myofibroblast differentiation is critical for proper skin wound healing. Neoexpression of α-smooth muscle actin (α-SMA), a marker for myofibroblast differentiation, is driven by transforming growth factor (TGF)-ß receptor-mediated signaling. Hyaluronan and its three synthesizing enzymes, hyaluronan synthases (Has 1, 2, and 3), also participate in this process. Closure of skin wounds is significantly accelerated in Has1/3 double-knockout (Has1/3-null) mice. Herein, TGF-ß activity and dermal collagen maturation were increased in Has1/3-null healing skin. Cultures of primary skin fibroblasts isolated from Has1/3-null mice had higher levels of TGF-ß activity, α-SMA expression, and phosphorylation of p38 mitogen-activated protein kinase at Thr180/Tyr182, compared with wild-type fibroblasts. p38α mitogen-activated protein kinase was a necessary element in a noncanonical TGF-ß receptor signaling pathway driving α-SMA expression in Has1/3-null fibroblasts. Myocardin-related transcription factor (MRTF), a cofactor that binds to the transcription factor serum response factor (SRF), was also critical. Nuclear localization of MRTF was increased, and MRTF binding to SRF was enhanced in Has1/3-null fibroblasts. Inhibition of MRTF or SRF expression by RNA interference suppresses α-SMA expression at baseline and diminished its overexpression in Has1/3-null fibroblasts. Interestingly, total matrix metalloproteinase activity was increased in healing skin and fibroblasts from Has1/3-null mice, possibly explaining the increased TGF-ß activation.

Interplay Between Chemotherapy-Activated Cancer Associated Fibroblasts and Cancer Initiating Cells Expressing CD44v6 Promotes Colon Cancer Resistance.

Cancer-initiating cells (CICs) drive colorectal tumor growth by their supportive niches where CICs interact with multiple cell types within the microenvironment, including cancer-associated fibroblasts (CAFs). We investigated the interplay between the CICs and the clinically relevant chemotherapeutic FOLFOX that creates the persistent tumorigenic properties of colorectal CICs, and stimulates the microenvironmental factors derived from the CAFs. We found that the CICs expressing an immunophenotype (CD44v6[+]) promote FOLFOX-resistance and that the CIC-immunophenotype was enhanced by factors secreted by CAFs after FOLFOX treatment These secreted factors included periostin, IL17A and WNT3A, which induced CD44v6 expression by activating WNT3A/ß-catenin signaling. Blocking the interaction between CICs with any of these CAF-derived factors through tissue-specific conditional silencing of CD44v6 significantly reduced colorectal tumorigenic potential. To achieve this, we generated two unique vectors (floxed-pSico-CD44v6 shRNA plus Fabpl-Cre) that were encapsulated into transferrin coated PEG-PEI/(nanoparticles), which when introduced in vivo reduced tumor growth more effectively than using CD44v6-blocking antibodies. Notably, this tissue-specific conditional silencing of CD44v6 resulted in long lasting effects on self-renewal and tumor growth associated with a positive feedback loop linking WNT3A signaling and alternative-splicing of CD44. These findings have crucial clinical implications suggesting that therapeutic approaches for modulating tumor growth that currently focus on cell-autonomous mechanisms may be too limited and need to be broadened to include mechanisms that recognize the interplay between the stromal factors and the subsequent CIC-immunophenotype enrichment. Thus, more specific therapeutic approaches may be required to block a chemotherapy induced remodeling of a microenvironment that acts as a paracrine regulator to enrich CD44v6 (+) in colorectal CICs.

Proteomic, mechanical, and biochemical development of tissue-engineered neocartilage.

BACKGROUND: The self-assembling process of cartilage tissue engineering is a promising technique to heal cartilage defects, preventing osteoarthritic changes. Given that chondrocytes dedifferentiate when expanded, it is not known if cellular expansion affects the development of self-assembled neocartilage. The objective of this study was to use proteomic, mechanical, and biochemical analyses to quantitatively investigate the development of self-assembled neocartilage derived from passaged, rejuvenated costal chondrocytes. METHODS: Yucatan minipig costal chondrocytes were used to create self-assembled neocartilage constructs. After 1, 4, 7, 14, 28, 56, or 84 days of self-assembly, constructs were analyzed through a variety of histological, biomechanical, biochemical, and proteomic techniques. RESULTS: It was found that temporal trends in neocartilage formation are similar to those seen in native hyaline articular cartilage development. For example, between days 7 and 84 of culture, tensile Young's modulus increased 4.4-times, total collagen increased 2.7-times, DNA content decreased 69.3%, collagen type II increased 1.5-times, and aggrecan dropped 55.3%, mirroring trends shown in native knee cartilage. Importantly, collagen type X, which is associated with cartilage calcification, remained at low levels (≤ 0.05%) at all neocartilage developmental time points, similar to knee cartilage (< 0.01%) and unlike donor rib cartilage (0.98%). CONCLUSIONS: In this work, bottom-up proteomics, a powerful tool to interrogate tissue composition, was used for the first time to quantify and compare the proteome of a developing engineered tissue to a recipient tissue. Furthermore, it was shown that self-assembled, costal chondrocyte-derived neocartilage is suitable for a non-homologous approach in the knee.

Proteomic, mechanical, and biochemical characterization of cartilage development.

The objective of this work is to examine the development of porcine cartilage by analyzing its mechanical properties, biochemical content, and proteomics at different developmental stages. Cartilage from the knees of fetal, neonatal, juvenile, and mature pigs was analyzed using histology, mechanical testing, biochemical assays, fluorophore-assisted carbohydrate electrophoresis, and bottom-up proteomics. Mature cartilage has 2.2-times the collagen per dry weight of fetal cartilage, and fetal cartilage has 2.1-times and 17.9-times the glycosaminoglycan and DNA per dry weight of mature cartilage, respectively. Tensile and compressive properties peak in the juvenile stage, with a tensile modulus 4.7-times that of neonatal. Proteomics analysis reveals increases in collagen types II and III, while collagen types IX, XI, and XIV, and aggrecan decrease with age. For example, collagen types IX and XI decrease 9.4-times and 5.1-times, respectively from fetal to mature. Mechanical and biochemical measurements have their greatest developmental changes between the neonatal and juvenile stages, where mechanotransduction plays a major role. Bottom-up proteomics serves as a powerful tool for tissue characterization, showing results beyond those of routine biochemical analysis. For example, proteomic analysis shows significant drops in collagen types IX, XI, and XIV throughout development, which shows insight into the permanence of cartilage's matrix. Changes in overall glycosaminoglycan content compared to aggrecan and link protein indicate non-enzymatic degradation of aggrecan structures or hyaluronan in mature cartilage. In addition to tissue characterization, bottom-up proteomics techniques are critical in tissue engineering efforts toward repair or regeneration of cartilage in animal models. STATEMENT OF SIGNIFICANCE: In this study, the development of porcine articular cartilage is interrogated through biomechanical, biochemical, and proteomic techniques, to determine how mechanics and extracellular matrix composition change from fetal to mature cartilage. For the first time, a bottom-up proteomics approach is used to reveal a wide variety of protein changes through aging; for example, the collagen subtype composition of the cartilage increases in collagen types II and III, and decreases in collagen types IX, XI, and XIV. This analysis shows that bottom-up proteomics is a critical tool in tissue characterization, especially toward developing a deeper understanding of matrix composition and development in tissue engineering studies.

Periostin/Filamin-A: A Candidate Central Regulatory Axis for Valve Fibrogenesis and Matrix Compaction.

BACKGROUND: Discoveries in the identification of transcription factors, growth factors and extracellular signaling molecules have led to the detection of downstream targets that modulate valvular tissue organization that occurs during development, aging, or disease. Among these, matricellular protein, periostin, and cytoskeletal protein filamin A are highly expressed in developing heart valves. The phenotype of periostin null indicates that periostin promotes migration, survival, and differentiation of valve interstitial cushion cells into fibroblastic lineages necessary for postnatal valve remodeling/maturation. Genetically inhibiting filamin A expression in valve interstitial cushion cells mirrored the phenotype of periostin nulls, suggesting a molecular interaction between these two proteins resulted in poorly remodeled valve leaflets that might be prone to myxomatous over time. We examined whether filamin A has a cross-talk with periostin/signaling that promotes remodeling of postnatal heart valves into mature leaflets. RESULTS: We have previously shown that periostin/integrin-ß1 regulates Pak1 activation; here, we revealed that the strong interaction between Pak1 and filamin A proteins was only observed after stimulation of VICs with periostin; suggesting that periostin/integrin-ß-mediated interaction between FLNA and Pak1 may have a functional role in vivo. We found that FLNA phosphorylation (S2152) is activated by Pak1, and this interaction was observed after stimulation with periostin/integrin-ß1/Cdc42/Rac1 signaling; consequently, FLNA binding to Pak1 stimulates its kinase activity. Patients with floppy and/or prolapsed mitral valves, when genetically screened, were found to have point mutations in the filamin A gene at P637Q and G288R. Expression of either of these filamin A mutants failed to increase the magnitude of filamin A (S2152) expression, Pak1-kinase activity, actin polymerization, and differentiation of VICs into mature mitral valve leaflets in response to periostin signaling. CONCLUSION: PN-stimulated bidirectional interaction between activated FLNA and Pak1 is essential for actin cytoskeletal reorganization and the differentiation of immature VICs into mature valve leaflets.

FOLFOX Therapy Induces Feedback Upregulation of CD44v6 through YB-1 to Maintain Stemness in Colon Initiating Cells.

Cancer initiating cells (CICs) drive tumor formation and drug-resistance, but how they develop drug-resistance characteristics is not well understood. In this study, we demonstrate that chemotherapeutic agent FOLFOX, commonly used for drug-resistant/metastatic colorectal cancer (CRC) treatment, induces overexpression of CD44v6, MDR1, and oncogenic transcription/translation factor Y-box-binding protein-1 (YB-1). Our study revealed that CD44v6, a receptor for hyaluronan, increased the YB-1 expression through PGE2/EP1-mTOR pathway. Deleting CD44v6, and YB-1 by the CRISPR/Cas9 system attenuates the in vitro and in vivo tumor growth of CICs from FOLFOX resistant cells. The results of DNA:CD44v6 immunoprecipitated complexes by ChIP (chromatin-immunoprecipitation) assay showed that CD44v6 maintained the stemness traits by promoting several antiapoptotic and stemness genes, including cyclin-D1, BCL2, FZD1, GINS-1, and MMP9. Further, computer-based analysis of the clones obtained from the DNA:CD44v6 complex revealed the presence of various consensus binding sites for core stemness-associated transcription factors "CTOS" (c-Myc, TWIST1, OCT4, and SOX2). Simultaneous expressions of CD44v6 and CTOS in CD44v6 knockout CICs reverted differentiated CD44v6-knockout CICs into CICs. Finally, this study for the first time describes a positive feedback loop that couples YB-1 induction and CD44 alternative splicing to sustain the MDR1 and CD44v6 expressions, and CD44v6 is required for the reversion of differentiated tumor cells into CICs.

Sirtuin 1 reduces hyaluronan synthase 2 expression by inhibiting nuclear translocation of NF-κB and expression of the long-noncoding RNA HAS2-AS1.

Hyaluronan (HA) is one of the most prevalent glycosaminoglycans of the vascular extracellular matrix (ECM). Abnormal HA accumulation within blood vessel walls is associated with tissue inflammation and is prominent in most vascular pathological conditions such as atherosclerosis and restenosis. Hyaluronan synthase 2 (HAS2) is the main hyaluronan synthase enzyme involved in HA synthesis and uses cytosolic UDP-glucuronic acid and UDP-GlcNAc as substrates. The synthesis of UDP-glucuronic acid can alter the NAD+/NADH ratio via the enzyme UDP-glucose dehydrogenase, which oxidizes the alcohol group at C6 to the COO- group. Here, we show that HAS2 expression can be modulated by sirtuin 1 (SIRT1), the master metabolic sensor of the cell, belonging to the class of NAD+-dependent deacetylases. Our results revealed the following. 1) Treatments of human aortic smooth muscle cells (AoSMCs) with SIRT1 activators (SRT1720 and resveratrol) inhibit both HAS2 expression and accumulation of pericellular HA coats. 2) Tumor necrosis factor α (TNFα) induced HA-mediated monocyte adhesion and AoSMC migration, whereas SIRT1 activation prevented immune cell recruitment and cell motility by reducing the expression levels of the receptor for HA-mediated motility, RHAMM, and the HA-binding protein TNF-stimulated gene 6 protein (TSG6). 3) SIRT1 activation prevented nuclear translocation of NF-κB (p65), which, in turn, reduced the levels of HAS2-AS1, a long-noncoding RNA that epigenetically controls HAS2 mRNA expression. In conclusion, we demonstrate that both HAS2 expression and HA accumulation by AoSMCs are down-regulated by the metabolic sensor SIRT1.

Hyaluronan Derived From the Limbus is a Key Regulator of Corneal Lymphangiogenesis.

Purpose: We recently reported that the glycosaminoglycan hyaluronan (HA), which promotes inflammatory angiogenesis in other vascular beds, is an abundant component of the limbal extracellular matrix. Consequently, we have explored the possibility that HA contributes to lymphangiogenesis in the inflamed cornea. Methods: To study the role of HA on lymphangiogenesis, we used mice lacking the hyaluronan synthases and injury models that induce lymphangiogenesis. Results: Here we report that HA regulates corneal lymphangiogenesis, both during post-natal development and in response to adult corneal injury. Furthermore, we show that injury to the cornea by alkali burn upregulates both HA production and lymphangiogenesis and that these processes are ablated in HA synthase 2 deficient mice. Conclusion: These findings raise the possibility that therapeutic blockade of HA-mediated lymphangiogenesis might prevent the corneal scarring and rejection that frequently results from corneal transplantation.

The journey of hyaluronan research in the Journal of Biological Chemistry.

Hyaluronan has a very simple structure. It is a linear glycosaminoglycan composed of disaccharide units of GlcNAc and d-glucuronic acid with alternating ß-1,4 and ß-1,3 glycosidic bonds that can be repeated 20,000 or more times, a molecular mass >8 million Da, and a length >20 µm. However, it has a very complex biology. It is a major, ubiquitous component of extracellular matrices involved in everything from fertilization, development, inflammations, to cancer. This JBC Review highlights some of these processes that were initiated through publications in the Journal of Biological Chemistry.

Periostin/ß1integrin interaction regulates p21-activated kinases in valvular interstitial cell survival and in actin cytoskeleton reorganization.

The matricellular protein periostin (PN) promotes postnatal valve remodeling and maturation. Incomplete remodeling of the valve can trigger degenerative processes that lead to a myxomatous phenotype that includes loss of PN. However, signaling pathways involved that link valvular-interstitial-fibroblast cells (VICs) to proliferation, migration and actin remodeling functions are unclear. The p21-activated kinases (Paks) have been shown to regulate cytoskeleton rearrangements and cell proliferation/adhesion/migration functions in a variety of cellular contexts, including normal cells and cancer cells. This study shows that Pak1, but not Pak2 and Pak4, is a critical mediator of VIC survival and actin organization, and that the molecular signaling regulating actin-remodeling is initiated upon PN/beta-integrin-induced phosphorylation of the focal-adhesion-kinase (Fak) (Y397). Molecular and pharmacological inhibition of key components of PN/Fak/Akt1 signaling abolished the PN-induced actin polymerization and the activation of mTOR, p70S6K and Pak1. Similarly, blocking mTOR inhibited p70S6K, Pak1 phosphorylation and consequently actin-polymerization. Accordingly, inhibiting p70S6K blocked Pak1 phosphorylation and actin polymerization, and subsequently inhibited adhesion and growth of VICs. Periostin-induced Akt1 activation of Pak1 is independent of Cdc42 and Rac1 GTPases, and Akt1 is both downstream and upstream of Pak1. Further, the PN-Pak1-induced Akt1 protects cells from apoptosis through suppression of transcriptional activation of Forkhead-Transcription-Factor (FKHR). In contrast, kinase deficient Pak1 increases apoptosis by increasing FKHR-mediated transcriptional activation. These studies define new functional significance of PN-Fak-Akt1-Pak1 signaling that at least partly regulates Akt1-induced actin polymerization and FKHR-mediated transcriptional activation, which may eventually regulate the mature-valve-leaflet remodeling function, and also FKHR-mediated transcriptional activation for pro-survival of VICs.

Heparin affects cytosolic glucose responses of hyperglycemic dividing mesangial cells.

Mesangial expansion underlies diabetic nephropathy, leading to sclerosis and renal failure. The glycosaminoglycan heparin inhibits mesangial cell growth, but the molecular mechanism is unclear. Here, rat mesangial cells (RMCs) were growth-arrested in the G0/G1 phase of cell division, stimulated to divide in normal glucose (5.6 mm) or high glucose (25.6 mm) with or without heparin, and analyzed for glucose uptake. We observed that RMCs entering the G1 phase in normal glucose with or without heparin rapidly cease glucose uptake. RMCs entering G1 in high glucose sustained glucose uptake for the first 3 h, and high-glucose exposure of RMCs only in the first 8 h of G1 induced the formation of an extracellular monocyte-adhesive hyaluronan matrix after cell division was completed. Moreover, a low heparin concentration under high-glucose conditions blocked glucose uptake by 1 h into G1 Of note, glucose transporter 4 (glut4) localized on the RMC surface at G0/G1 and was internalized into G1 cells under normal glucose conditions with or without heparin within 30 min. We also noted that, under high-glucose conditions, glut4 remained on the RMC surface for at least 2 h into G1 and was internalized by 4 h without heparin and within 1 h with heparin. These results provide evidence that the influx of glucose in hyperglycemic dividing RMCs initiates intermediate glucose metabolism, leading to increased cytosolic UDP sugars, and induces abnormal intracellular hyaluronan synthesis during the S phase of cell division.

Midgut Laterality Is Driven by Hyaluronan on the Right.

For many years, biologists have focused on the role of Pitx2, expressed on the left side of developing embryos, in governing organ laterality. Here, we identify a different pathway during left-right asymmetry initiated by the right side of the embryo. Surprisingly, this conserved mechanism is orchestrated by the extracellular glycosaminoglycan, hyaluronan (HA) and is independent of Pitx2 on the left. Whereas HA is normally synthesized bilaterally as a simple polysaccharide, we show that covalent modification of HA by the enzyme Tsg6 on the right triggers distinct cell behavior necessary to drive the conserved midgut rotation and to pattern gut vasculature. HA disruption in chicken and Tsg6-/- mice results in failure to initiate midgut rotation and perturbs vascular development predisposing to midgut volvulus. Our study leads us to revise the current symmetry-breaking paradigm in vertebrates and demonstrates how enzymatic modification of HA matrices can execute the blueprint of organ laterality.

Variation in primary and culture-expanded cells derived from connective tissue progenitors in human bone marrow space, bone trabecular surface and adipose tissue.

BACKGROUND AIMS: Connective tissue progenitors (CTPs) embody the heterogeneous stem and progenitor cell populations present in native tissue. CTPs are essential to the formation and remodeling of connective tissue and represent key targets for tissue-engineering and cell-based therapies. To better understand and characterize CTPs, we aimed to compare the (i) concentration and prevalence, (ii) early in vitro biological behavior and (iii) expression of surface-markers and transcription factors among cells derived from marrow space (MS), trabecular surface (TS), and adipose tissues (AT). METHODS: Cancellous-bone and subcutaneous-adipose tissues were collected from 8 patients. Cells were isolated and cultured. Colony formation was assayed using Colonyze software based on ASTM standards. Cell concentration ([Cell]), CTP concentration ([CTP]) and CTP prevalence (PCTP) were determined. Attributes of culture-expanded cells were compared based on (i) effective proliferation rate and (ii) expression of surface-markers CD73, CD90, CD105, SSEA-4, SSEA-3, SSEA-1/CD15, Cripto-1, E-Cadherin/CD324, Ep-CAM/CD326, CD146, hyaluronan and transcription factors Oct3/4, Sox-2 and Nanog using flow cytometry. RESULTS: Mean [Cell], [CTP] and PCTP were significantly different between MS and TS samples (P = 0.03, P = 0.008 and P= 0.0003), respectively. AT-derived cells generated the highest mean total cell yield at day 6 of culture-4-fold greater than TS and more than 40-fold greater than MS per million cells plated. TS colonies grew with higher mean density than MS colonies (290 ± 11 versus 150 ± 11 cell per mm2; P = 0.0002). Expression of classical-mesenchymal stromal cell (MSC) markers was consistently recorded (>95%) from all tissue sources, whereas all the other markers were highly variable. CONCLUSIONS: The prevalence and biological potential of CTPs are different between patients and tissue sources and lack variation in classical MSC markers. Other markers are more likely to discriminate differences between cell populations in biological performance. Understanding the underlying reasons for variation in the concentration, prevalence, marker expression and biological potential of CTPs between patients and source tissues and determining the means of managing this variation will contribute to the rational development of cell-based clinical diagnostics and targeted cell-based therapies.

Quantification of hyaluronan (HA) using a simplified fluorophore-assisted carbohydrate electrophoresis (FACE) procedure.

Hyaluronan (HA) exhibits numerous important roles in physiology and pathologies, and these facts necessitate an ability to accurately and reproducibly measure its quantities in tissues and cell cultures. Our group previously reported a rigorous and analytical procedure to quantify HA (and chondroitin sulfate, CS) using a reductive amination chemistry and separation of the fluorophore-conjugated, unsaturated disaccharides unique to HA and CS on high concentration acrylamide gels. This procedure is known as fluorophore-assisted carbohydrate electrophoresis (FACE) and has been adapted for the detection and quantification of all glycosaminoglycan types. While this previous FACE procedure is relatively straightforward to implement by carbohydrate research investigators, many nonglycoscience laboratories now studying HA biology might have difficulties establishing this prior FACE procedure as a routine assay for HA. To address this need, we have greatly simplified our prior FACE procedure for accurate and reproducible assessment of HA in tissues and cell cultures. This chapter describes in detail this simplified FACE procedure and, because it uses an enzyme that degrades both HA and CS, investigators will also gain additional insight into the quantities of CS in the same samples dedicated for HA analysis.

Transforming growth factor ß1 (TGFß1)-induced CD44V6-NOX4 signaling in pathogenesis of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive clinical syndrome of fatal outcome. The lack of information about the signaling pathways that sustain fibrosis and the myofibroblast phenotype has prevented the development of targeted therapies for IPF. Our previous study showed that isolated fibrogenic lung fibroblasts have high endogenous levels of the hyaluronan receptor, CD44V6 (CD44 variant containing exon 6), which enhances the TGFß1 autocrine signaling and induces fibroblasts to transdifferentiate into myofibroblasts. NADPH oxidase 4 (NOX4) enzyme, which catalyzes the reduction of O2 to hydrogen peroxide (H2O2), has been implicated in the cardiac and lung myofibroblast phenotype. However, whether CD44V6 regulates NOX4 to mediate tissue repair and fibrogenesis is not well-defined. The present study assessed the mechanism of how TGF-ß-1-induced CD44V6 regulates the NOX4/reactive oxygen species (ROS) signaling that mediates the myofibroblast differentiation. Specifically, we found that NOX4/ROS regulates hyaluronan synthesis and the transcription of CD44V6 via an effect upon AP-1 activity. Further, CD44V6 is part of a positive-feedback loop with TGFß1/TGFßRI signaling that acts to increase NOX4/ROS production, which is required for myofibroblast differentiation, myofibroblast differentiation, myofibroblast extracellular matrix production, myofibroblast invasion, and myofibroblast contractility. Both NOX4 and CD44v6 are up-regulated in the lungs of mice subjected to experimental lung injury and in cases of human IPF. Genetic (CD44v6 shRNA) or a small molecule inhibitor (CD44v6 peptide) targeting of CD44v6 abrogates fibrogenesis in murine models of lung injury. These studies support a function for CD44V6 in lung fibrosis and offer proof of concept for therapeutic targeting of CD44V6 in lung fibrosis disorders.