In Vitro Study of Cytotoxic Mechanisms of Alkylphospholipids and Alkyltriazoles in Acute Lymphoblastic Leukemia Models.

This study investigates the efficacy of miltefosine, alkylphospholipid, and alkyltriazolederivative compounds against leukemia lineages. The cytotoxic effects and cellular and molecular mechanisms of the compounds were investigated. The inhibitory potential and mechanism of inhibition of cathepsins B and L, molecular docking simulation, molecular dynamics and binding free energy evaluation were performed to determine the interaction of cathepsins and compounds. Among the 21 compounds tested, C9 and C21 mainly showed cytotoxic effects in Jurkat and CCRF-CEM cells, two human acute lymphoblastic leukemia (ALL) lineages. Activation of induced cell death by C9 and C21 with apoptotic and necrosis-like characteristics was observed, including an increase in annexin-V+propidium iodide-, annexin-V+propidium iodide+, cleaved caspase 3 and PARP, cytochrome c release, and nuclear alterations. Bax inhibitor, Z-VAD-FMK, pepstatin, and necrostatin partially reduced cell death, suggesting that involvement of the caspase-dependent and -independent mechanisms is related to cell type. Compounds C9 and C21 inhibited cathepsin L by a noncompetitive mechanism, and cathepsin B by a competitive and noncompetitive mechanism, respectively. Complexes cathepsin-C9 and cathepsin-C21 exhibited significant hydrophobic interactions, water bridges, and hydrogen bonds. In conclusion, alkyltriazoles present cytotoxic activity against acute lymphoblastic lineages and represent a promising scaffold for the development of molecules for this application.

The role of heparan sulphate in development: the ectodermal story.

Heparan sulphate (HS) is ubiquitously expressed and is formed of repeating glucosamine and glucuronic/iduronic acid units which are generally highly sulphated. HS is found in tissues bound to proteins forming HS proteoglycans (HSPGs) which are present on the cell membrane or in the extracellular matrix. HSPGs influence a variety of biological processes by interacting with physiologically important proteins, such as morphogens, creating storage pools, generating morphogen gradients and directly mediating signalling pathways, thereby playing vital roles during development. This review discusses the vital role HS plays in the development of tissues from the ectodermal lineage. The ectodermal layer differentiates to form the nervous system (including the spine, peripheral nerves and brain), eye, epidermis, skin appendages and tooth enamel.

Heparan sulfate regulates hair follicle and sebaceous gland morphogenesis and homeostasis.

Hair follicle (HF) morphogenesis and cycling are a result of intricate autonomous epithelial-mesenchymal interactions. Once the first HF cycle is complete it repeatedly undergoes cyclic transformations. Heparan sulfate (HS) proteoglycans are found on the cell surface and in the extracellular matrix where they influence a variety of biological processes by interacting with physiologically important proteins, such as growth factors. Inhibition of heparanase (an HS endoglycosidase) in in vitro cultured HFs has been shown to induce a catagen-like process. Therefore, this study aimed to elucidate the precise role of HS in HF morphogenesis and cycling. An inducible tetratransgenic mouse model was generated to excise exostosin glycosyltransferase 1 (Ext1) in keratin 14-positive cells from P21. Interestingly, EXT1(StEpiΔ/StEpiΔ) mice presented solely anagen HFs. Moreover, waxing the fur to synchronize the HFs revealed accelerated hair regrowth in the EXT1(StEpiΔ/StEpiΔ) mice and hindered cycling into catagen. The ablation of HS in the interfollicular epidermal cells of mature skin led to the spontaneous formation of new HFs and an increase in Sonic Hedgehog expression resembling wild-type mice at P0, thereby indicating that the HS/Sonic Hedgehog signaling pathway regulates HF formation during embryogenesis and prevents HF formation in mature skin. Finally, the knock-out of HS also led to the morphogenesis and hyperplasia of sebaceous glands and sweat glands in mature mice, leading to exacerbated sebum production and accumulation on the skin surface. Therefore, our findings clearly show that an intricate control of HS levels is required for HF, sebaceous gland, and sweat gland morphogenesis and HF cycling.

Umbilical cord mesenchymal stem cells suppress host rejection: the role of the glycocalyx.

Umbilical cord mesenchymal stem cells (UMSCs) have unique immunosuppressive properties enabling them to evade host rejection and making them valuable tools for cell therapy. We previously showed that human UMSCs survive xenograft transplantation and successfully correct the corneal clouding defects associated with the mouse model for the congenital metabolic disorder mucopolysaccharidosis VII. However, the precise mechanism by which UMSCs suppress the immune system remains elusive. This study aimed to determine the key components involved in the ability of the UMSCs to modulate the inflammatory system and to identify the inflammatory cells that are regulated by the UMSCs. Our results show that human UMSCs transplanted into the mouse stroma 24 h after an alkali burn suppress the severe inflammatory response and enable the recovery of corneal transparency within 2 weeks. Furthermore, we demonstrated in vitro that UMSCs inhibit the adhesion and invasion of inflammatory cells and also the polarization of M1 macrophages. UMSCs also induced the maturation of T-regulatory cells and led to inflammatory cell death. Moreover, UMSCs exposed to inflammatory cells synthesize a rich extracellular glycocalyx composed of the chondroitin sulfate-proteoglycan versican bound to a heavy chain (HC)-modified hyaluronan (HA) matrix (HC-HA). This matrix also contains TNFα-stimulated gene 6 (TSG6), the enzyme that transfers HCs to HA, and pentraxin-3, which further stabilizes the matrix. Our results, both in vivo and in vitro, show that this glycocalyx confers the ability for UMSCs to survive the host immune system and to regulate the inflammatory cells.

Mesenchymal stem cells for treating ocular surface diseases.

Mesenchymal stem cells (MSC) have become a promising tool for cell therapy in regenerative medicine. They are readily available, demonstrate powerful differentiation capabilities and present immunosuppressive properties that aid them in surviving from host immune rejection for its great potential use in allograft. Currently clinical trials are underway using MSC, both culture-expanded allogeneic and autologous, for the treatment of a range of diseases not treatable by conventional therapies. A vast array of studies has dedicated towards the use of MSC for treating corneal diseases with very promising outcomes. MSC have successfully differentiated into keratocytes both in vitro and in vivo, and corneal epithelial cells in vitro, but it is uncertain if MSC can assume corneal epithelial cells in vivo. However, to date few studies have unequivocally established the efficacy of MSC for treating corneal endothelial defects. Currently, the diversity in protocols of the isolation and expansion of MSC are hindering to the assessment of cell treatment ability and the further development of treatment regimens. Therefore, future studies should develop international standards for MSC isolation and characterization. In this review, we discuss recent advances in MSC for treating ocular surface diseases.

Transplantation of human umbilical mesenchymal stem cells cures the corneal defects of mucopolysaccharidosis VII mice.

Mucopolysaccharidosis (MPS) are a family of related disorders caused by a mutation in one of the lysosomal exoglycosidases which leads to the accumulation of glycosaminoglycans (GAGs). MPS VII, caused by a mutation in ß-glucuronidase, manifests hepatomegaly, skeletal dysplasia, short stature, corneal clouding, and developmental delay. Current treatment regimens for MPS are not effective for treating corneal clouding and impaired mental development. We hypothesized that human umbilical mesenchymal stem cells (UMSCs) transplanted into the corneal stroma could participate in the catabolism of GAGs providing a means of cell therapy for MPS. For such treatment, human UMSCs were intrastromally transplanted into corneas of MPS VII mice. UMSC transplantation restored the dendritic and hexagonal morphology of host keratocytes and endothelial cells, respectively, and in vivo confocal microscopy (HRT-II) revealed reduced corneal haze. Immunohistochemistry using antibodies against heparan sulfate and chondroitin sulfate chains as well as lysosomal-associated membrane protein 2 revealed a decrease in GAG content and both lysosomal number and size in the treated corneas. Labeling UMSC intracellular compartments prior to transplantation revealed the distribution of UMSC vesicles throughout the corneal stroma and endothelium. An in vitro coculture assay between skin fibroblasts isolated from MPS VII mice and UMSC demonstrated that neutral vesicles released by the UMSC are taken up by the fibroblasts and proceed to fuse with the acidic lysosomes. Therefore, transplanted UMSCs participate both in extracellular GAG turnover and enable host keratocytes to catabolize accumulated GAG products, suggesting that UMSC could be a novel alternative for treating corneal defects associated with MPS and other congenital metabolic disorders.

Enterolobium contortisiliquum trypsin inhibitor (EcTI), a plant proteinase inhibitor, decreases in vitro cell adhesion and invasion by inhibition of Src protein-focal adhesion kinase (FAK) signaling pathways.

Tumor cell invasion is vital for cancer progression and metastasis. Adhesion, migration, and degradation of the extracellular matrix are important events involved in the establishment of cancer cells at a new site, and therefore molecular targets are sought to inhibit such processes. The effect of a plant proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on the adhesion, migration, and invasion of gastric cancer cells was the focus of this study. EcTI showed no effect on the proliferation of gastric cancer cells or fibroblasts but inhibited the adhesion, migration, and cell invasion of gastric cancer cells; however, EcTI had no effect upon the adhesion of fibroblasts. EcTI was shown to decrease the expression and disrupt the cellular organization of molecules involved in the formation and maturation of invadopodia, such as integrin ß1, cortactin, neuronal Wiskott-Aldrich syndrome protein, membrane type 1 metalloprotease, and metalloproteinase-2. Moreover, gastric cancer cells treated with EcTI presented a significant decrease in intracellular phosphorylated Src and focal adhesion kinase, integrin-dependent cell signaling components. Together, these results indicate that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathways.

A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model.

Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution.

Structural Determinants of Substrate Recognition and Catalysis by Heparan Sulfate Sulfotransferases.

Heparan sulfate (HS) and heparin contain imprinted "sulfation codes", which dictate their diverse physiological and pathological functions. A group of orchestrated biosynthetic enzymes cooperate in polymerizing and modifying HS chains. The biotechnological development of enzymes that can recreate this sulfation pattern on synthetic heparin is challenging, primarily due to the paucity of quantitative data for sulfotransferase enzymes. Herein, we identified critical structural characteristics that determine substrate specificity and shed light on the catalytic mechanism of sugar sulfation of two HS sulfotransferases, 2-O-sulfotransferase (HS2ST) and 6-O-sulfotransferase (HS6ST). Two sets of molecular clamps in HS2ST recognize appropriate substrates; these clamps flank the acceptor binding site on opposite sides. The hexuronic epimers, and not their puckers, have a critical influence on HS2ST selectivity. In contrast, HS6ST recognizes a broader range of substrates. This promiscuity is granted by a conserved tryptophan residue, W210, that positions the acceptor within the active site for catalysis by means of strong electrostatic interactions. Lysines K131 and K132 act in concert with a second tryptophan, W153, shedding water molecules from within the active site, thus providing HS6ST with a binding preference toward 2-O-sulfated substrates. QM/MM calculations provided valuable mechanistic insights into the catalytic process, identifying that the sulfation of both HS2ST and HS6ST follows a SN2-like mechanism. When they are taken together, our findings reveal the molecular basis of how these enzymes recognize different substrates and catalyze sugar sulfation, enabling the generation of enzymes that could create specific heparin epitopes.

TRPV channels mediate temperature-sensing in human corneal endothelial cells.

The physiology and transparency of the cornea are dependent on corneal endothelial function. The role of temperature sensitive ion channels in maintaining such activity is unknown. This study was undertaken to probe for the functional expression of such pathways in human corneal endothelial cells (HCEC). We used HCEC-12, an immortalized population derived from whole corneal endothelium, and two morphologically distinct clonal cell lines derived from HCEC-12 (HCEC-H9C1, HCEC-B4G12) to probe for gene expression and function of transient receptor potential (TRP) channels of the vanilloid (V) isoform subfamily (i.e. TRPV1-3) in these cell types. Expression of TRPV isotypes 1, 2 and 3 were detected by RT-PCR. Protein expression of TRPV1 in situ was confirmed by immunostaining of corneoscleral remnants after keratoplasty. TRPV1-3 functional activity was evident based on capsaicin-induced Ca(2+) transients and induction of these responses through rises in ambient temperature from 25 degrees C to over 40 degrees C. The currents underlying Ca(2+) transients were characterized with a novel high throughput patch-clamp system. The TRPV1 selective agonist, capsaicin (CAP) (10-20 microM) increased non-selective cation whole-cell currents resulting in calcium increases that were fully blocked by either the TRPV1 antagonist capsazepine (CPZ) or removal of extracellular calcium. Similarly, heating from room temperature to over 40 degrees C increased the same currents resulting in calcium increases that were significantly reduced by the TRP channel blockers lanthanum chloride (La(3+)) (100 microM) and ruthenium-red (RuR) (10 microM), respectively. Moreover, application of the TRPV channel opener 2-aminoethoxydiphenyl borate (2-APB) (400 microM) led to a reversible increase in intracellular Ca(2+) indicating putative TRPV1-3 channel activity. Taken together, TRPV activity modulation by temperature underlies essential homeostatic mechanisms contributing to the support of corneal endothelial function under different ambient conditions.

Analysis of Cancer Stromal Reaction Using an O-ring Co-culture Assay.

We have developed a 2D heterotypic co-culture technique between fibroblasts and cancer cells that enables the study of the stromal reaction. For such, stromal cells are seeded and cultured immediately around a tumour cell line, and the cells establish cell-cell contacts, as well as a gradient of soluble factors throughout the stromal cells, similar to that found in tissues. Thus, this system also enables the researcher to distinguish between events that are caused by direct cell-cell contact and secreted factors.

3D Stroma Invasion Assay.

We have developed a 3D co-culture system composed of fibroblasts and colorectal cancer cells that enables us to study the desmoplastic reaction. This method also enables us to study the influence of the desmoplastic reaction on the migration of colorectal cancer cells through the surrounding stroma. This protocol has been previously published (Coulson- Thomas et al., 2011 ) and is described here in more detail.

Isolation and Analysis of Proteoglycans and Glycosaminoglycans from Archaeological Bones and Teeth.

We have developed methods for isolating proteoglycans and glycosaminoglycans from archaeological bones and teeth. These methods have been previously published (Coulson- Thomas et al., 2015 ) and are described here in more detail. In the case of glycosaminoglycans, the method was a previously described method ( Nader et al., 1999 ) which we optimized for archeological samples.

The role of proteoglycans in the reactive stroma on tumor growth and progression.

The stroma surrounding tumors can either restrict or promote tumor growth and progression, and both the cellular and non-cellular components of the stroma play an active role. The cellular components in the surrounding stroma include tumor-associated fibroblasts, host tissue cells and immune cells. The non-cellular components, which form the extracellular matrix (ECM) scaffold, include proteoglycans, collagen, proteinases, growth factors and cytokines. For tumorigenesis to occur it is necessary for tumor cells to modify the surrounding stroma. Tumor cells have mechanisms for achieving this, such as co-opting fibroblasts and modifying the ECM they produce, degrading the surrounding ECM and/or synthesizing a favorable ECM to support invasion. Proteoglycans are an important component of the ECM and play an active role in tumor growth and progression. The expression and glycosylation patterns of proteoglycans are altered in the stroma surrounding tumors and these molecules may support or restrict tumor growth and progression depending on the type and stage of tumor. In the present review we discuss the difference between the tumor promoting and restricting stromal reactions surrounding tumors and the role proteoglycans play.

Loss of corneal epithelial heparan sulfate leads to corneal degeneration and impaired wound healing.

PURPOSE: Heparan sulfate (HS) is a highly modified glycosaminoglycan (GAG) bound to a core protein to form heparan sulfate proteoglycans (HSPGs) that are vital in many cellular processes ranging from development to adult physiology, as well as in disease, through interactions with various protein ligands. This study aimed to elucidate the role of HS in corneal epithelial homeostasis and wound healing. METHODS: An inducible quadruple transgenic mouse model was generated to excise Ext1 and Ndst1, which encode the critical HS chain elongation enzyme and N-deacetylase/N-sulfotransferase, respectively, in keratin 14-positive cells upon doxycycline induction. RESULTS: EXT(Δ/ΔCEpi) mice (deletion of Ext1 in corneal epithelium) induced at P20 presented progressive thinning of the corneal epithelium with a significant loss in the number of epithelial layers by P55. EXT(Δ/ΔCEpi) mice presented tight junction disruption, loss of cell-basement membrane adhesion complexes, and impaired wound healing. Interestingly, EXT(Δ/ΔCEpi) and NDST(Δ/ΔCEpi) mice presented an increase in cell proliferation, which was assayed by both Ki67 staining and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Moreover, EXT(Δ/ΔCEpi) mice presented compromised epithelial stratification 7 days after a debridement wound. The conditional knockout of HS from keratocytes using the keratocan promoter led to no corneal abnormalities or any disruption in wound healing. CONCLUSIONS: Corneal epithelial cells require HS for maintaining corneal homeostasis, and the loss of epithelial HS leads to both impaired wound healing and impaired corneal stratification.

Insights into the N-sulfation mechanism: molecular dynamics simulations of the N-sulfotransferase domain of NDST1 and mutants.

Sulfation patterns along glycosaminoglycan (GAG) chains dictate their functional role. The N-deacetylase N-sulfotransferase family (NDST) catalyzes the initial downstream modification of heparan sulfate and heparin chains by removing acetyl groups from subsets of N-acetylglucosamine units and, subsequently, sulfating the residual free amino groups. These enzymes transfer the sulfuryl group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS), yielding sulfated sugar chains and 3'-phosphoadenosine-5'-phosphate (PAP). For the N-sulfotransferase domain of NDST1, Lys833 has been implicated to play a role in holding the substrate glycan moiety close to the PAPS cofactor. Additionally, Lys833 together with His716 interact with the sulfonate group, stabilizing the transition state. Such a role seems to be shared by Lys614 through donation of a proton to the bridging oxygen of the cofactor, thereby acting as a catalytic acid. However, the relevance of these boundary residues at the hydrophobic cleft is still unclear. Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive. In this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics. In addition, the binding location of the glycan moiety, PAPS and PAP within the active site of NDST1 throughout the sulfate transfer were determined by intermediate state analysis. Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.

Lumican binds ALK5 to promote epithelium wound healing.

Lumican (Lum), a small leucine-rich proteoglycan (SLRP) family member, has multiple matricellular functions both as an extracellular matrix component and as a matrikine regulating cell proliferation, gene expression and wound healing. To date, no cell surface receptor has been identified to mediate the matrikine functions of Lum. This study aimed to identify a perspective receptor that mediates Lum effects on promoting wound healing. Transforming growth factor-ß receptor 1 (ALK5) was identified as a potential Lum-interacting protein through in silico molecular docking and molecular dynamics. This finding was verified by biochemical pull-down assays. Moreover, the Lum function on wound healing was abrogated by an ALK5-specific chemical inhibitor as well as by ALK5 shRNAi. Finally, we demonstrated that eukaryote-specific post-translational modifications are not required for the wound healing activity of Lum, as recombinant GST-Lum fusion proteins purified from E. coli and a chemically synthesized LumC13 peptide (the last C-terminal 13 amino acids of Lum) have similar effects on wound healing in vitro and in vivo.