Efficient Photodynamic Therapy of Prostate Cancer Cells through an Improved Targeting of the Cation-Independent Mannose 6-Phosphate Receptor.

The aim of the present work is the development of highly efficient targeting molecules to specifically address mesoporous silica nanoparticles (MSNs) designed for the photodynamic therapy (PDT) of prostate cancer. We chose the strategy to develop a novel compound that allows the improvement of the targeting of the cation-independent mannose 6-phosphate receptor, which is overexpressed in prostate cancer. This original sugar, a dimannoside-carboxylate (M6C-Man) grafted on the surface of MSN for PDT applications, leads to a higher endocytosis and thus increases the efficacy of MSNs.

Modulation of Melanotransferrin and Transferrin Receptor 1 (TFRC)- and CD44-based Signaling for TFRC Up-regulation in Human Melanoma Cells.

BACKGROUND: The human melanoma cell line IGR-1 was used for the detection and regulation of both melanotransferrin (MTf) and transferrin receptor 1 (TFRC, CD71). While the function in iron transport of the TFRC is well documented the functional importance of MTf is not yet fully understood. Due to the up-regulation of TFRC by hyaluronan (HA) some components and aspects of CD44 signaling were investigated. MATERIALS AND METHODS: The cell-surface proteins MTf, TFRC and ERBB2 receptor tyrosine kinase 2 (ERBB2) were detected by immunoluminescent technique using different polyclonal and monoclonal antibodies. Ionomycin was used to inhibit ß-catenin/T-cell-specific transcription factor (TCF) association, essential in HA-CD44-ERBB2 signaling. RESULTS: MTf, was found to be resistant to phosphatidylinositol-specific phospholipase C. However, MTf as well as TFRC were sensitive to partial proteolytic degradation by pronase E and trypsin. The expression of MTf was shown to be up-regulated by mannose-6-phosphate and that of TFRC by HA. Ionomycin at 10 µM inhibited TFRC up-regulation. However, at 50 µM it induced a 7.5-fold increase of TFRC concentration. CONCLUSION: Our results suggest that human melanoma cells are able to up-regulate TFRC expression using HA/CD44 signaling. The whole pathway comprises of the sequence: HA/CD44, neural Wiskott-Aldrich syndrome protein (N-WASP), ERBB2, ß-catenin/TCF, c-MYC and TFRC. Since ß-catenin is also known to be a component of wingless/Int-1-Frizzled signaling that also leads to transcriptional c-MYC activation, the pathway found here might be alternatively used by melanoma cells for iron supply, necessary for cell proliferation.

Inhibition of insulin-like growth factor II (IGF-II)-dependent cell growth by multidentate pentamannosyl 6-phosphate-based ligands targeting the mannose 6-phosphate/IGF-II receptor.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) binds M6P-capped ligands and IGF-II at different binding sites within the ectodomain and mediates ligand internalization and trafficking to the lysosome. Multivalent M6P-based ligands can cross-bridge the M6P/IGF2R, which increases the rate of receptor internalization, permitting IGF-II binding as a passenger ligand and subsequent trafficking to the lysosome, where the IGF-II is degraded. This unique feature of the receptor may be exploited to design novel therapeutic agents against IGF-II-dependent cancers that will lead to decreased bioavailable IGF-II within the tumor microenvironment. We have designed a panel of M6P-based ligands that bind to the M6P/IGF2R with high affinity in a bivalent manner and cause decreased cell viability. We present evidence that our ligands bind through the M6P-binding sites of the receptor and facilitate internalization and degradation of IGF-II from conditioned medium to mediate this cellular response. To our knowledge, this is the first panel of synthetic bivalent ligands for the M6P/IGF2R that can take advantage of the ligand-receptor interactions of the M6P/IGF2R to provide proof-of-principle evidence for the feasibility of novel chemotherapeutic agents that decrease IGF-II-dependent growth of cancer cells.

Design of Potent Mannose 6-Phosphate Analogues for the Functionalization of Lysosomal Enzymes To Improve the Treatment of Pompe Disease.

Improving therapeutics delivery in enzyme replacement therapy (ERT) for lysosomal storage disorders is a challenge. Herein, we present the synthesis of novel analogues of mannose 6-phosphate (M6P), known as AMFAs and functionalized at the anomeric position for enzyme grafting. AMFAs are non-phosphate serum-resistant derivatives that efficiently bind the cation-independent mannose 6-phosphate receptor (CI-M6PR), which is the main pathway to address enzymes to lysosomes. One of the AMFAs was used to improve the treatment of the lysosomal myopathy Pompe disease, in which acid α-glucosidase (GAA) is defective. AMFA grafting on a M6P-free recombinant GAA led to a higher uptake of the GAA in adult Pompe fibroblasts in culture as compared to Myozyme, the M6P recombinant GAA. Moreover, the treatment of Pompe adult mice with the AMFA-grafted recombinant enzyme led to a remarkable improvement, even at low doses, in muscle functionality and regeneration, whereas Myozyme had limited efficacy.

Soluble DPP-4 up-regulates toll-like receptors and augments inflammatory reactions, which are ameliorated by vildagliptin or mannose-6-phosphate.

OBJECTIVE: Studies have shown that dipeptidyl peptidase-4 (DPP-4) inhibitors have anti-inflammatory effects. Soluble DPP-4 (sDPP-4) has been considered as an adipokine of which actions need to be further characterized. METHODS: We investigated the pro-inflammatory actions of sDPP-4 and the anti-inflammatory effects of DPP-4 inhibition, using vildagliptin, as an enzymatic inhibitor, and mannose-6-phosphate (M6P) as a competitive binding inhibitor. RESULTS: In lipopolysaccharide (LPS)-stimulated RAW264.7 cells, vildagliptin suppressed the increased expression of inducible nitric oxide synthase (iNOS) and phosphorylated JNK (pJNK), activation of the NF-κB pathway, and the resultant NO and proinflammatory cytokine production. Although sDPP-4 alone did not affect the protein level of iNOS or pJNK or the production of NO in RAW264.7 cells, it did amplify iNOS expression, NO responses, and proinflammatory cytokine production in LPS-stimulated RAW264 cells. As a probable mechanism, we found that sDPP-4 caused dose-dependent increases in the expression levels of toll-like receptor 4 (TLR4) and TLR2 in RAW264.7 cells, and that these alterations were inhibited by vildagliptin, M6P, or bisindolylmaleimide II, a protein kinase C inhibitor. Either vildagliptin or M6P suppressed iNOS expression and NO and cytokine production in LPS+DPP-4-co-stimulated macrophages, while combined treatment of the co-stimulated cells with both agents had increased anti-inflammatory effects compared with either treatment alone. Intravenous injection of sDPP-4 to C57BL/6J mice increased the expression of both TLRs in kidney and white adipose tissues. CONCLUSION: Our findings suggest that sDPP-4 enhances inflammatory actions via TLR pathway, while DPP-4 inhibition with either an enzymatic or binding inhibitor has anti-inflammatory effects.

New mannose derivatives: The tetrazole analogue of mannose-6-phosphate as angiogenesis inhibitor.

Two novel compounds with mannose-derived structure, bearing a tetrazole (compound 3) and a sulfone group (compound 4) in terminal position, have been prepared from methyl α-d-mannopyranoside in reduced number of steps. The angiogenic activity of 3 and 4 has been screened using the chick chorioallantoic membrane (CAM) method. Tetrazole 3 has been identified to possess a promising bioactivity, being identified as angiogenesis inhibitor, with 68% of neovascular vessels when compared to control (PBS).

Mannose-6-phosphate facilitates early peripheral nerve regeneration in thy-1-YFP-H mice.

The formation of scar tissue following nerve injury has been shown to adversely affect nerve regeneration and evidence suggests that mannose-6-phosphate (M6P), a potential scar reducing agent that affects transforming growth factor (TGF)-ß activation, may enhance nerve regeneration. In this study we utilized thy-1-YFP-H mice - a transgenic strain expressing yellow fluorescent protein (YFP) within a subset of axons - to enable visual analysis of axons regenerating through a nerve graft. Using this strain of mouse we have developed analysis techniques to visualize and quantify regeneration of individual axons across the injury site following the application of either M6P or vehicle to the site of nerve injury. No significant differences were found in the proportion of axons regenerating through the graft between M6P- and vehicle-treated grafts at any point along the graft length. Maximal sprouting occurred at 1.0mm from the proximal graft ending in both groups. The maximum change in sprouting levels for both treatment groups occurred between the graft start and 0.5-mm interval for both treatment groups. The difference between repair groups was significant at this point with a greater increase seen in the vehicle group than the M6P group. The average length of axons regenerating across the initial graft entry was significantly shorter in M6P- than in vehicle-treated grafts, indicating that they encountered less impedance. Application of M6P appears to reduce the disruption of regenerating axons and may therefore facilitate quicker recovery; this is likely to result from altered scar tissue formation in M6P grafts in the early stages of recovery. This study also establishes the usefulness of our methods of analysis using the thy-1-YFP-H mouse strain to visualize and quantify regeneration at the level of the individual axon.

Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors.

Bioreactor process changes can have a profound effect on the yield and quality of biotechnology products. Mannose-6-phosphate (M6P) glycan content and the enzymatic catalytic kinetic parameters are critical quality attributes (CQAs) of many therapeutic enzymes used to treat lysosomal storage diseases (LSDs). Here, we have evaluated the effect of adding butyrate to bioreactor production cultures of human recombinant ß-glucuronidase produced from CHO-K1 cells, with an emphasis on CQAs. The ß-glucuronidase produced in parallel bioreactors was quantified by capillary electrophoresis, the catalytic kinetic parameters were measured using steady-state analysis, and mannose-6-phosphorylation status was assessed using an M6P-specific single-chain antibody fragment. Using this approach, we found that butyrate treatment increased ß-glucuronidase production up to approximately threefold without significantly affecting the catalytic properties of the enzyme. However, M6P content in ß-glucuronidase was inversely correlated with the increased enzyme production induced by butyrate treatment. This assessment demonstrated that although butyrate dramatically increased ß-glucuronidase production in bioreactors, it adversely impacted the mannose-6-phosphorylation of this LSD therapeutic enzyme. This strategy may have utility in evaluating manufacturing process changes to improve therapeutic enzyme yields and CQAs.

Histomorphometric changes in repaired mouse sciatic nerves are unaffected by the application of a scar-reducing agent.

Microsurgical repair of transected peripheral nerves is compromised by the formation of scar tissue and the development of a neuroma, thereby limiting the success of regeneration. The aim of this study was to quantify histomorphometrically the structural changes in neural tissue that result from repair, and determine the effect of mannose-6-phosphate (M6P), a scar-reducing agent previously shown to enhance regeneration. In anaesthetised C57-black-6 mice, the left sciatic nerve was sectioned and repaired using four epineurial sutures. Either 100 µL of 600 mm M6P (five animals) or 100 µL of phosphate-buffered saline (placebo controls, five animals) was injected into and around the nerve repair site. A further group acted as sham-operated controls. After recovery for 6 weeks, the nerve was harvested for analysis using light and electron microscopy. Analysis revealed that when compared with sham controls, myelinated axons had smaller diameters both proximal and distal to the repair. Myelinated axon counts, axonal density and size all decreased across the repair site. There were normal numbers and densities of non-myelinated axons both proximal and distal to the repair. However, there were more Remak bundles distal to the repair site, and fewer non-myelinated axons per Remak bundle. Application of M6P did not affect any of these parameters.

Mannose-6-phosphate regulates destruction of lipid-linked oligosaccharides.

Mannose-6-phosphate (M6P) is an essential precursor for mannosyl glycoconjugates, including lipid-linked oligosaccharides (LLO; glucose(3)mannose(9)GlcNAc(2)-P-P-dolichol) used for protein N-glycosylation. In permeabilized mammalian cells, M6P also causes specific LLO cleavage. However, the context and purpose of this paradoxical reaction are unknown. In this study, we used intact mouse embryonic fibroblasts to show that endoplasmic reticulum (ER) stress elevates M6P concentrations, leading to cleavage of the LLO pyrophosphate linkage with recovery of its lipid and lumenal glycan components. We demonstrate that this M6P originates from glycogen, with glycogenolysis activated by the kinase domain of the stress sensor IRE1-α. The apparent futility of M6P causing destruction of its LLO product was resolved by experiments with another stress sensor, PKR-like ER kinase (PERK), which attenuates translation. PERK's reduction of N-glycoprotein synthesis (which consumes LLOs) stabilized steady-state LLO levels despite continuous LLO destruction. However, infection with herpes simplex virus 1, an N-glycoprotein-bearing pathogen that impairs PERK signaling, not only caused LLO destruction but depleted LLO levels as well. In conclusion, the common metabolite M6P is also part of a novel mammalian stress-signaling pathway, responding to viral stress by depleting host LLOs required for N-glycosylation of virus-associated polypeptides. Apparently conserved throughout evolution, LLO destruction may be a response to a variety of environmental stresses.

Annexin VI is a mannose-6-phosphate-independent endocytic receptor for bovine ß-glucuronidase.

Endocytosis and transport of bovine liver ß-glucuronidase to lysosomes in human fibroblasts are mediated by two receptors: the well-characterized cation-independent mannose 6-phosphate receptor (IGF-II/Man6PR) and an IGF-II/Man6PR-independent receptor, which recognizes a Ser-Trp*-Ser sequence present on the ligand. The latter receptor was detergent extracted from bovine liver membranes and purified. LC/ESI-MS/MS analysis revealed that this endocytic receptor was annexin VI (AnxA6). Several approaches were used to confirm this finding. First, the binding of bovine ß-glucuronidase to the purified receptor from bovine liver membranes and His-tagged recombinant human AnxA6 protein was confirmed using ligand-blotting assays. Second, western blot analysis using antibodies raised against IGF-II/Man6PR-independent receptor as well as commercial antibodies against AnxA6 confirmed that the receptor and AnxA6 were indeed the same protein. Third, double immunofluorescence experiments in human fibroblasts confirmed a complete colocalization of the bovine ß-glucuronidase and the AnxA6 receptor on the plasma membrane. Lastly, two cell lines were stably transfected with a plasmid containing the cDNA for human AnxA6. In both transfected cell lines, an increase in cell surface AnxA6 and in mannose 6-phosphate-independent endocytosis of bovine ß-glucuronidase was detected. These results indicate that AnxA6 is a novel receptor that mediates the endocytosis of the bovine ß-glucuronidase.

The effect of Mannose-6-Phosphate on recovery after sciatic nerve repair.

We have determined the effect of applying Mannose-6-Phosphate (M6P), a scar reducing agent, to a site of sciatic nerve repair. In anaesthetised C57-Black-6 mice, the left sciatic nerve was sectioned and repaired using 4 epineurial sutures. Either 100 µl of 600 mM Mannose-6-Phosphate (29 animals), or 100 µl of phosphate buffered saline as a placebo control (29 animals), was injected into and around the nerve repair site. A further group acted as sham-operated controls. After 6 or 12 weeks of recovery the extent of regeneration was assessed electrophysiologically and the percentage area of collagen staining at the repair site was analysed using picrosirius red and image analysis. Gait analysis was undertaken pre-operatively and at 1, 3, 6, 9 and 12 weeks postoperatively, to assess functional recovery. At 6 weeks the compound action potentials recorded from the regenerated nerves in the M6P group were significantly larger than in the placebo controls (P=0.015), and the conduction velocities were significantly faster (P=0.005), but there were no significant differences between these groups at 12 weeks. Gait analysis suggested better early functional recovery in the M6P group. In both repair groups there was a significant reduction in collagen staining between 6 and 12 weeks, suggestive of scar remodelling. We conclude that the normal scar remodelling process aids long term recovery in repaired nerves. Administration of 600 mM M6P to the nerve repair site enhances nerve regeneration and functional recovery in the early stages, and may lead to improved outcomes.

Cation-independent mannose 6-phosphate receptor inhibitor (PXS25) inhibits fibrosis in human proximal tubular cells by inhibiting conversion of latent to active TGF-beta1.

Hyperglycemia and hypoxia have independent and convergent roles in the development of renal disease. Transforming growth factor-ß(1) (TGF-ß(1)) is a key cytokine promoting the production of extracellular matrix proteins. The cationic-independent mannose 6-phosphate receptor (CI-M6PR) is a membrane protein that binds M6P-containing proteins. A key role is to activate latent TGF-ß(1). PXS25, a novel CI-MPR inhibitor, has antifibrotic properties in skin fibroblasts, but its role in renal fibrosis is unclear. The aim was to study the role of PXS25 in matrix protein production under high glucose ± hypoxic conditions in human proximal tubule (HK-2) cells. HK-2 cells were exposed to high glucose (30 mM) ± 100 µM PXS25 in both normoxic (20% O(2)) and hypoxic (1% O(2)) conditions for 72 h. Cellular fibronectin, collagen IV, and matrix metalloproteinase-2 (MMP-2) and MMP-9 were assessed. Total and active TGF-ß(1) were measured by ELISA. High glucose and hypoxia independently induced TGF-ß(1) production. Active TGF-ß(1), but not total TGF-ß(1) was reduced with concurrent PXS25 in the presence of high glucose, but not in hyperglycemia+hypoxia conditions. Hyperglycemia induced fibronectin and collagen IV production (P < 0.05), as did hypoxia, but only hyperglycemia-induced increases in matrix proteins were suppressed by concurrent PXS25 exposure. High glucose induced MMP-2 and -9 in normoxic and hypoxic conditions, which was not modified in the presence of PXS25. High glucose and hypoxia can independently induce endogenous active TGF-ß(1) production in human proximal tubular cells. PXS25 inhibits conversion of high glucose-induced release of active TGF-ß(1), only in the absence of hypoxia.

A comparison between the effects of three potential scar-reducing agents applied at a site of sciatic nerve repair.

We have investigated the effect of three potential scar-reducing agents applied at a sciatic nerve repair site in C57-black-6 mice. Under anaesthesia the nerve was transected, repaired using four epineurial sutures, and 100 µl of either triamcinolone acetonide (1 mg/100 µl), an interleukin-10 peptide fragment (125 ng/100 µl or 500 ng/100 µl) or mannose-6-phosphate (M6P, 200 mM or 600 mM) was injected into and around the nerve. After 6 weeks the extent of regeneration was assessed electrophysiologically by determining the ratio of the compound action potential (CAP) modulus evoked by electrical stimulation of the nerve 2 mm distal or proximal to the repair site. The conduction velocity of the fastest components in the CAP was also calculated. The percentage area of collagen staining (PAS) at the repair site was analysed using Picrosirius Red and image analysis. Comparisons were made with a placebo group (100 µl of phosphate buffered saline) and sham-operated controls. The median CAP modulus ratio in the 600 mM M6P group was 0.44, which was significantly higher than in the placebo group (0.24, P=0.012: Kruskal-Wallis test). Conduction velocities were also faster in the 600 mM M6P group (median 30 m s(-1)) than in the placebo group (median 27.8 m s(-1); P=0.0197: Kruskal-Wallis test). None of the other treated groups were significantly different from the placebo, and all had significantly lower CAP ratios than the sham controls (P<0.05). All repair groups had a significantly higher PAS for collagen than sham controls. We conclude that the administration of 600 mM mannose-6-phosphate to a nerve repair site enhances axonal regeneration.

Inhibition effect of mannose-6-phosphate on expression of transforming growth factor Beta receptor in flexor tendon cells.

Transforming growth factor beta (TGF-ß) has an important role in tendon healing and adhesion formation. Inhibiting TGF-ß and its receptor expression may prevent adhesions after tendon open. The goal of this study was to examine the effects of mannose-6-phosphate, a natural inhibitor of TGF-ß, on TGF-ß and its receptor production in tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes of rabbit flexor tendons. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendons and cultured separately. The cells were divided into 2 groups at random: an experiment group supplemented with mannose-6-phosphate and a control group without mannose-6-phosphate. The expression of TGF-ß and TGF-ß receptor was quantified with enzyme-linked immunosorbent assay. The luciferase assay measured TGF-ß bioactivity. Transforming growth factor beta expression in the experimental group was not decreased compared with the control group, with no significant difference (P>.05) Transforming growth factor beta receptor expression in the experiment group was significantly lower than that in control group (P<.05). Mannose-6-phosphate significantly decreased the expression of TGF-ß receptor and TGF-ß bioactivity. Modulation of mannose-6-phosphate levels may provide a means of modulating the effects of TGF-ß on adhesion formation in flexor tendon wound healing.

Inhibition of TGF-ß-induced collagen production in rabbit flexor tendon with mannose-6-phosphate in vitro.

The flexor tendon affects postoperative range of motion in the hand. Transforming growth factor-beta (TGF-ß) is a key cytokine in the adhesion formation between the flexor tendon and its surrounding fibro-osseous sheath. The purpose of this study was to examine the inhibition of TGF-ß-induced collagen-I production in rabbit flexor tendons with mannose-6-phosphate in vitro. Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes from rabbit flexor tendons were isolated and each was supplemented with TGF-ß along with increasing doses of mannose-6-phosphate. The enzyme-linked immunosorbent assay (ELISA) and reverse-transcription polymerase chain reaction (RT-PCR) measured collagen-I production. The luciferase assay measured TGF-ß bioactivity. Results were compared with TGF-ß alone and unsupplemented controls. TGF-ß-induced collagen-I production was downregulated significantly with the addition of mannose-6-phosphate in a dose-dependent manner in all 3 cells cultures. The mannose-6-phosphate also reduced TGF-ß bioactivity. The study shows that mannose-6-phosphate was effective in TGF-ß inhibition in cultured flexor tendon cells. The findings presented here encourage further experiments that use the agents to modulate TGF-ß levels and reduce adhesion formation after flexor tendon repair.

Inhibition of transforming growth factor-beta1 and its effects on human corneal fibroblasts by mannose-6-phosphate Potential for preventing haze after refractive surgery.

PURPOSE: To evaluate the action of mannose-6-phosphate (M6P) as an inhibitor of transforming growth factor-beta1 (TGF-beta1) and its effects on human corneal fibroblasts. SETTING: Department of Academic Ophthalmology, Rayne Institute, St. Thomas' Hospital, London, United Kingdom. METHOD: Keratocytes were cultured in serum-containing medium with added TGF-beta1 and in serum-containing medium containing TGF-beta1 with M6P. Controls consisted of cells in serum-containing medium alone and cells cultured in serum-containing medium with M6P. Differentiation of fibroblasts into myofibroblasts was detected by immunohistochemistry for alpha-smooth muscle actin. Images were captured by laser confocal microscopy. RESULTS: The differentiation of cells into myofibroblasts was significantly greater after culture in medium containing TGF-beta1 than in control cultures. Myofibroblast differentiation of cells was significantly reduced when cultured in medium containing TGF-beta1 with M6P (P<.001). CONCLUSIONS: Mannose-6-phosphate significantly reduced TGF-beta1-mediated transformation of human corneal fibroblasts into myofibroblasts; it is a potential modulator of corneal wound healing and may reduce haze after refractive surgery. FINANCIAL DISCLOSURE: Neither author has a financial or proprietary interest in any material or method mentioned.

[Effects of mannose-6-phosphate on transforming growth factor beta and transforming growth factor beta receptor expression of flexor tendon cells].

OBJECTIVE: By culturing tendon sheath fibroblasts, epitenon tenocytes and endotenon tenocytes of rabbits' tendon in vitro, to study the effects of mannose-6-phosphate on transforming growth factor beta (TGF-beta) peptide and receptor expression, and to provide the experimental basis for preventing the tendon healing adhesion by mannose-6-phosphate. METHODS: Eight adult New Zealand white rabbits, regardless of their gender and weighing 4.0-4.5 kg, were selected. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendon and cultured separately. All 3 cells were divided into 2 groups at random after cells were adjusted to a concentration of 4 x 10(4) per well and 1 x 10(4)/mL. The first was the control group without supplementation. The experimental group was supplemented with mannose-6-phosphate. The expressions of TGF-beta and TGF-beta receptor were quantified with enzyme-linked immunosorbent assay. The expression of TGF-beta1 mRNA was also assessed with in situ hybridization and the expression of TGF-beta1 was assessed with immunohistochemistry. RESULTS: The expressions of TGF-beta and TGF-beta receptor in experimental group were significantly lower than that in control group (P < 0.05). The expression levels of TGF-beta1 and TGF-beta2 decreased in descending order of tendon sheath fibroblasts (36.1%, 37.9%), epitenon tenocytes (31.0%, 32.1%), and endotenon tenocytes (31.2%, 27.0%). The expression levels of TGF-beta3 decreased in descending order of endotenon tenocytes (42.5%), tendon sheath fibroblasts (41.2%), and epitenon tenocytes (33.3%). The expression levels of TGF-beta receptor 1 and TGF-beta receptor 2 decreased in descending order of epitenon tenocytes (29.9%, 26.2%), endotenon tenocytes (27.8%, 23.5%), and tendon sheath fibroblasts (23.1%, 20.0%). The expression levels of TGF-beta receptor 3 decreased in descending order of endotenon tenocytes (26.1%), epitenon tenocytes (19.2%), and tendon sheath fibroblasts (15.8%). In experimental group, the positive expression of TGF-beta1 mRNA and the expression level of intracellular TGF-beta1 mRNA in all 3 tendon cells were significantly lower than those in the control group (P < 0.05). Immunohistochemical staining showed the expressions of TGF-beta1 in all 3 tendon cells were significantly lower in the experimental group than in the control group. CONCLUSION: Mannose-6-phosphate can significantly decrease the expressions of TGF-beta peptide, TGF-beta receptor, and TGF-beta1 mRNA. Modulation of mannose-6-phosphate levels may provide a mean of modulating the effects of TGF-beta on adhesion formation in flexor tendon wound healing.

Synthesis and evaluation of non-hydrolyzable D-mannose 6-phosphate surrogates reveal 6-deoxy-6-dicarboxymethyl-D-mannose as a new strong inhibitor of phosphomannose isomerases.

Non-hydrolyzable d-mannose 6-phosphate analogues in which the phosphate group was replaced by a phosphonomethyl, a dicarboxymethyl, or a carboxymethyl group were synthesized and kinetically evaluated as substrate analogues acting as potential inhibitors of type I phosphomannose isomerases (PMIs) from Saccharomyces cerevisiae and Escherichia coli. While 6-deoxy-6-phosphonomethyl-d-mannose and 6-deoxy-6-carboxymethyl-D-mannose did not inhibit the enzymes significantly, 6-deoxy-6-dicarboxymethyl-D-mannose appeared as a new strong competitive inhibitor of both S. cerevisiae and E. coli PMIs with K(m)/K(i) ratios of 28 and 8, respectively. We thus report the first malonate-based inhibitor of an aldose-ketose isomerase to date. Phosphonomethyl mimics of the 1,2-cis-enediolate high-energy intermediate postulated for the isomerization reaction catalyzed by PMIs were also synthesized but behave as poor inhibitors of PMIs. A polarizable molecular mechanics (SIBFA) study was performed on the complexes of d-mannose 6-phosphate and two of its analogues with PMI from Candida albicans, an enzyme involved in yeast infection homologous to S. cerevisiae and E. coli PMIs. It shows that effective binding to the catalytic site occurs with retention of the Zn(II)-bound water molecule. Thus the binding of the hydroxyl group on C1 of the ligand to Zn(II) should be water-mediated. The kinetic study reported here also suggests the dianionic character of the phosphate surrogate as a likely essential parameter for strong binding of the inhibitor to the enzyme active site.