Restoration of the antigen (Ag)-specific immune tolerance in an allergic environment is refractory. B cells are involved in immune regulation. Whether B cells facilitate the generation of Ag-specific immune tolerance in an allergic environment requires further investigation. This paper aims to elucidate the mechanism by which B cells restore the Ag-specific immune tolerance in an allergic environment. In this study, a B cell-deficient mouse model was created by injecting an anti-CD20 antibody. The frequency of tolerogenic dendritic cell (TolDC) was assessed by flow cytometry. The levels of cytokines were determined by enzyme-linked immunosorbent assay. The expression of thrombospondin-1 (TSP1) was assessed by quantitative real-time RT-PCR, Western blotting, and methylation-specific PCR. The results showed that B cells were required in the generation of the TGF-β-producing TolDCs in mice. B cell-derived TSP1 converted the latent TGF-β to the active TGF-β in DCs, which generated TGF-β-producing TolDCs. Exposure to IL-13 inhibited the expression of TSP1 in B cells by enhancing the TSP1 gene DNA methylation. Treating food allergy mice with Ag-specific immunotherapy and IL-13 antagonists restored the generation of TolDCs and enhanced the effect of specific immunotherapy. In conclusion, B cells play a critical role in the restoration of specific immune tolerance in an allergic environment. Blocking IL-13 in an allergic environment facilitated the generation of TolDCs and enhanced the therapeutic effect of immunotherapy.

OBJECTIVE

To test the hypothesis that primary open-angle glaucoma (POAG) patients have a systemic elevation of transforming growth factor β1 (TGFβ1).

METHODS

Plasma was prepared from blood samples drawn from patients of the Vanderbilt Eye Institute during clinic visits. Concentrations of total TGFβ1 and thrombospondin-1 (TSP1) in plasma were determined by ELISA. Statistical significance of differences between POAG and control samples was evaluated by Mann-Whitney test. Regression analysis was used to evaluate correlations between plasma TGFβ1 and patient age and between plasma TGFβ1 and TSP1.

RESULTS

Plasma samples were obtained from 148 POAG patients and 150 controls. Concentration of total TGFβ1 in the plasma of POAG patients (median = 3.25 ng/mL) was significantly higher (P < 0.0001) than in controls (median = 2.46 ng/mL). Plasma TGFβ1 was not correlated with age of patient (P = 0.17). Thrombospondin-1 concentration was also significantly higher (P < 0.0001) in POAG patients (median = 0.774 μg/mL) as compared to controls (median = 0.567 μg/mL). Plasma total TGFβ1 and TSP1 concentrations were linearly correlated (P < 0.0001).

CONCLUSIONS

Plasma samples from POAG patients display elevated total TGFβ1 compared to controls, consistent with elevated systemic TGFβ1 in POAG patients.

Histone deacetylase (HDAC) inhibitors have been reported to inhibit angiogenesis as well as tumor growth. Thrombospondin-1 (TSP1) has been recognized as a potent inhibitor of angiogenesis. Such an action of TSP1 may account for the effect of HDAC inhibitors. In the present study, we investigated the molecular mechanism by which trichostatin A, a HDAC inhibitor, induces the expression of TSP1 gene. Trichostatin A increased both mRNA and protein levels of TSP1 in HeLa cells. Promoter and actinomycin D chase assays showed that trichostatin A-induced TSP1 expression was regulated at the transcriptional level without changing mRNA stability. CCAAT box on the TSP1 promoter was found to primarily mediate the trichostatin A response by deletion and mutation analyses of the TSP1 promoter. Electrophoretic mobility shift assay indicated that CCAAT-binding factor (CBF) was specifically bound to the CCAAT box of TSP1 promoter. Moreover, chromatin immunoprecipitation assay showed that trichostatin A increased the binding of acetylated form of histone H3 to the CCAAT box region of TSP1 promoter. Taken together, these results strongly suggest that trichostatin A activates the transcription of TSP1 gene through the binding of transcription factor CBF to CCAAT box and the enhanced histone acetylation. Thus, the present study provides the clue that the inhibition of angiogenesis by trichostatin A is accomplished through the upregulation of TSP1, the anti-angiogenic factor.

To evaluate the predictive value of genes involved in resistance to platinum-taxane chemotherapy in patients with epithelial ovarian cancer (EOC). Microdissected formalin-fixed tumoral samples from 187 EOC patients' primary tumors (90 and 97 samples from matched patients in the experimental and validation sets, respectively) were analyzed. All specimens were analyzed for ATP7b, BRCA1, BRCA2, PARP1, UIMC1(RAP80), HOXA9, DAXX, TXN (TRX1), THBS1 (TSP1) and PRR13 (TXR1) mRNA expression by quantitative real-time PCR. Most of the patients (172 out of 187) received front-line carboplatin-paclitaxel regimen. Expression levels were correlated with overall (OS) and progression-free (PFS) survival by multivariate analysis. Patients with high TXN and THBS1 expression presented longer PFS (P=0.001 and P<0.001, respectively) and OS (P=0.024 and P<0.001, respectively). High TXR1 expression was associated with decreased PFS (P<0.001) and OS (P<0.001). Multivariate analysis demonstrated that high PRR13/low THBS1 expression was an independent factor for decreased PFS (hazards ratio: 1.94; 95% confidence interval (CI): 1.48-2.92; P=0.008) and OS (hazard ratio: 3.89; 95% CI: 2.16-6.87; P<0.001), whereas low TXN expression was correlated with decreased PFS (hazard ratio: 1.44; 95% CI: 1.05-2.84; P=0.043) and OS (hazard ratio: 2.38; 95% CI: 1.78-2.77; P=0.009). These findings indicate that PRR13/THBS1 and TXN expression could be used for the prediction of resistance to treatment of EOC patients and, therefore, merit to be further evaluated.

Angiogenesis plays an important role in the initiation and progression of many malignancies including prostate cancer (PCa). Therefore, genes implicated in angiogenic pathways could be susceptibility candidate genes for this malignancy. In this respect, we investigated the impact of functional genetic variants of TSP1 (N700S) and MMP9 (-1562 C/T) genes on the development and progression of PCa. This case-control study included 101 PCa patients and 106 healthy controls analyzed by polymerase chain reaction -restriction fragment length polymorphism assay. No association was observed between any of the TSP1 genotypes and PCa risk or severity; however, subjects carrying one copy of the MMP9 T allele exhibited threefold higher risk of developing PCa (OR = 2.86; P = 0.004). Regarding prognostic value, a significant association was found between the occurrence of the MMP9 T allele and the high-grade tumor (OR = 3.21; P = 0.004) and the advanced disease (OR = 2.47; P = 0.026). We also analyzed the effect of the combined genotypes on PCa risk. The patients with two high-risk genotypes exhibited 2.8-fold higher risk of developing PCa than those with only low-risk genotypes, but the association was not statistically significant. These findings suggest that MMP9 polymorphism is an independent risk factor of PCa development and aggressiveness.

Senescence is a tumor-suppressive mechanism induced by telomere shortening, oncogenes, or chemotherapy treatment. Although it is clear that this suppressive pathway leads to a permanent arrest in primary cells, this might not be the case in cancer cells that have inactivated their suppressive pathways. We have recently shown that subpopulations of cells can escape chemotherapy-mediated senescence and emerge as more transformed cells that induce tumor formation, resist anoikis, and are more invasive. In this study, we characterized this emergence and showed that senescent cells favor tumor growth and metastasis, in vitro and in vivo. Senescence escape was regulated by secreted proteins produced during emergence. Among these, we identified thrombospondin-1 (TSP1), a protein produced by senescent cells that prevented senescence escape. Using SWATH quantitative proteomic analysis, we found that TSP1 can be detected in the serum of patients suffering from triple-negative breast cancer and that its low expression was associated with treatment failure. The results also indicate that senescence escape is explained by the emergence of CD47^low cells that express a reduced level of CD47, the TSP1 receptor. The results show that CD47 expression is regulated by p21waf1. The cell cycle inhibitor was sufficient to maintain senescence since its downregulation in senescent cells increased cell emergence. This leads to the upregulation of Myc, which then binds to the CD47 promoter to repress its expression, allowing the generation of CD47^low cells that escape the suppressive arrest. Altogether, these results uncovered a new function for TSP1 and CD47 in the control of chemotherapy-mediated senescence.

Increasing evidence supports that the epithelial to mesenchymal transition (EMT) in breast cancer cells generates tumor initiating cells (TICs) but the contribution of the tumor microenvironment to these programs needs further elucidation. CCN6 (WISP3) is a secreted matrix-associated protein (36.9 kDa) of the CCN family (named after CTGF, Cyr61 and Nov) that is reduced or lost in invasive carcinomas of the breast with lymph node metastasis and in inflammatory breast cancer. CCN6 exerts breast cancer growth and invasion inhibitory functions, but the mechanisms remain to be defined. In the present study we discovered that ectopic CCN6 overexpression in triple negative (TN) breast cancer cells and in cells derived from patients is sufficient to induce a mesenchymal to epithelial transition (MET) and to reduce TICs. In vivo, CCN6 overexpression in the TIC population of MDA-MB-231 cells delayed tumor initiation, reduced tumor volume, and inhibited the development of metastasis. Our studies reveal a novel CCN6/Slug signaling axis that regulates Notch1 signaling activation, epithelial cell phenotype and breast TICs, which requires the conserved thrombospondin type 1 (TSP1) motif of CCN6. The relevance of these data to human breast cancer is highlighted by the finding that CCN6 protein levels are inversely correlated with Notch1 intracellular activated form (NICD1) in 69.5% of invasive breast carcinomas. These results demonstrate that CCN6 regulates epithelial and mesenchymal states transition and TIC programs, and pinpoint one responsible mechanism.

Thrombospondin 1 (TSP1) has been suggested as a counter receptor to platelet glycoprotein Ibα that supports initial platelet adhesion in absence of von Willebrand factor (VWF). Conversely, several other studies have shown that TSP1 interacts with VWF and may play a mechanistic role in modulating thrombosis. However, the in vivo evidence to support this mechanism remains unclear. Using intravital microscopy, in a 10% FeCl3-induced thrombosis model, we report similar platelet adhesion in Tsp1(-/-)/Vwf(-/-) mice compared with littermate Vwf(-/-) mice, suggesting that TSP1 does not mediate initial platelet adhesion in the absence of VWF. Tsp1(-/-) mice exhibited prolonged occlusion time and a significant decrease in the rate of thrombus growth (P < .05 vs wild-type), but not in the initial platelet adhesion. Complete deficiency of VWF abrogated the rate of thrombus growth in Tsp1(-/-) mice; therefore, we generated Tsp1(-/-)/Vwf(+/-) mice to determine whether TSP1 modulates thrombus growth under conditions of partial VWF deficiency. Tsp1(-/-)/Vwf(+/-) mice exhibited delayed thrombus growth kinetics and prolonged occlusion time (P < .05 vs Vwf(+/-)). Finally, we demonstrate that platelet-derived TSP1 modulates arterial thrombosis in vivo. We conclude that TSP1 released from platelets plays a mechanistic role in modulating thrombosis in the presence of VWF.

OBJECTIVE

Stromal vascularization is a frequent occurrence in herpes simplex keratitis (HSK) and carries a poor prognosis for penetrating keratoplasty. The pathogenesis may involve disruption of the normal equilibrium between angiogenic and anti-angiogenic factors in and around the cornea. Thrombospondin (TSP) 1 and 2 are multifunctional matricellular glycoproteins with potent anti-angiogenic properties and are expressed by human keratocytes in a stromal wound repair model. We hypothesize that the synthesis of these anti-angiogenic proteins by keratocytes is inhibited by HSV1 and that such a mechanism may contribute to stromal vascularization in HSK.

METHODS

Nonconfluent monolayers of human keratocytes were infected with HSV1 at a multiplicity of infection of 5 virus particles/keratocyte. Expression of TSP1 and TSP2 was determined by immunohistochemistry and SDS-polyacrylamide gel electrophoresis at 0, 2, 4, 6, 8, 24, 48, and 72 h after infection (ai). Expression of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) served as a control. Expression of immediate early and late viral proteins was also determined. Protein expression was quantified by densitometric analysis of the immunoblot bands.

RESULTS

Human keratocytes supported the growth of HSV1 at all times ai. TSP1 and TSP2 were downregulated as early as 4 h ai to a 50% reduction by 8 h (p<0.002), and were absent from 24 h ai (p<0.001). There was no change in the level of expression of GAPDH throughout the duration of the experiment. Immediate early viral proteins (HSV1:ICP27) could be detected from 6 h ai reaching maximum intensity 24 h ai and late proteins (HSV:1gD) were expressed from 24 h.

CONCLUSIONS

The synthesis of TSP1 and TSP2 is selectively downregulated by HSV1 infection in human keratocytes. Addition of these proteins or their angio-active peptides in early stage HSK therapy may be an important adjuvant in controlling HSV1 induced corneal vascularization.

BACKGROUND

Angiogenesis is regulated by a balance of both angiogenic inducers and inhibitors. This study was designed to evaluate the effect of both maximum-tolerated doses (MTD) and low-dose metronomic chemotherapy (LDM) on serum vascular endothelial growth factor (VEGF), thrombospondin-1 (TSP1) and VEGFR1 concentrations in patients with advanced nonsmall cell lung cancer.

METHODS

Forty consecutive patients with advanced stage nonsmall cell lung cancer were included in this prospective study. Twenty patients received MTD chemotherapy including 75 mg/m2 of cisplatin and 75 mg/m2 of docetaxel on day 1. The LDM treatment consisted of cisplatin 25 mg/m2 and docetaxel 25 mg/m2 were given to other 20 patients on weeks 1, 2 and 3. Serum levels were prospectively measured in serum by ELISA at four times; before chemotherapy and at 1, 2 and 3 weeks following initiation of chemotherapy.

RESULTS

The major finding in this study that MTD chemotherapy but not LDM chemotherapy resulted in significant changes in VEGFR1 and TSP1 serum levels. Due to the effect of LDM chemotherapy, we showed no statistically significant change in patients for all serum VEGF, TSP1 and VEGFR1 levels. Similarly, serum VEGF levels did not also change under MTD chemotherapy. The MTD chemotherapy induced significant and long-lasting increase of TSP1 levels and decrease of VEGFR1 levels that persisted for at least 3 weeks after the chemotherapy initiation. No significant correlations were found between serum VEGF and TSP1 levels in cancer patients treated with both LDM and MTD chemotherapy. The circulating angiogenic balance (TSP1/VEGF) is decreased in cancer patients (P=0.039).

CONCLUSIONS

The continuous/metronomic chemotherapy may not achieve a more pronounced antiangiogenic effect than MTD-scheduling chemotherapy. Future studies involving a larger number of patients are needed to confirm the present findings.

Bacteriophages recognize their host cells with the help of tail fiber and tailspike proteins that bind, cleave, or modify certain structures on the cell surface. The spectrum of ligands to which the tail fibers and tailspikes can bind is the primary determinant of the host range. Bacteriophages with multiple tailspike/tail fibers are thought to have a wider host range than their less endowed relatives but the function of these proteins remains poorly understood. Here, we describe the structure, function, and substrate specificity of three tailspike proteins of bacteriophage CBA120-TSP2, TSP3 and TSP4 (orf211 through orf213, respectively). We show that tailspikes TSP2, TSP3 and TSP4 are hydrolases that digest the O157, O77, and O78 Escherichia coli O-antigens, respectively. We demonstrate that recognition of the E. coli O157:H7 host by CBA120 involves binding to and digesting the O157 O-antigen by TSP2. We report the crystal structure of TSP2 in complex with a repeating unit of the O157 O-antigen. We demonstrate that according to the specificity of its tailspikes TSP2, TSP3, and TSP4, CBA120 can infect E. coli O157, O77, and O78, respectively. We also show that CBA120 infects Salmonella enterica serovar Minnesota, and this host range expansion is likely due to the function of TSP1. Finally, we describe the assembly pathway and the architecture of the TSP1-TSP2-TSP3-TSP4 branched complex in CBA120 and its related ViI-like phages.

To investigate inner retinal oxygen metabolic rate (IRMRO2) during early stages of type 1 diabetes in a transgenic mouse model.

In current study, we involved seven diabetic mice (Akita/+, TSP1-/-) and seven control mice (TSP1-/-), and applied visible-light optical coherence tomography (vis-OCT) to image functional parameters including retinal blood flow rate, oxygen saturation (sO2) and the IRMRO2 value longitudinally from 5 weeks of age to 13 weeks of age. After imaging at 13 weeks of age, we analyzed the imaging results, and examined histology of mouse retina.

Between diabetic mice and the control group, we observed significant differences in venous sO2 from 9 weeks of age (P = 0.006), and significant increment in IRMRO2 from 11 weeks of age (P = 0.001) in diabetic mice compared with control group. We did not find significant differences in retinal blood flow rate as well as arterial sO2 during imaging between diabetic and control mice. Histologic examination of diabetic and control mice at 13 weeks of age also revealed no anatomical retinal alternations.

In diabetic retinopathy, complications in retinal oxygen metabolism may occur before changes of retinal anatomical structure.

Thrombospondins (TSPs) constitute a family of secreted extracellular matrix proteins that have been shown to be involved in the formation of synapses in the central nervous system. In this study, we show that TSP1 and TSP2 are expressed in the cochlea, and offer the first description of their putative roles in afferent synapse development and function in the inner ear. We examined mice with deletions of TSP1, TSP2 and both (TSP1/TSP2) for inner ear development and function. Immunostaining for synaptic markers indicated a significant decrease in the number of formed afferent synapses in the cochleae of TSP2 and TSP1/TSP2 knockout (KO) mice at postnatal day (P)29. In functional studies, TSP2 and TSP1/TSP2 KO mice showed elevated auditory brainstem response (ABR) thresholds as compared with wild-type littermates, starting at P15, with the most severe phenotype being seen for TSP1/TSP2 KO mice. TSP1/TSP2 KO mice also showed reduced wave I amplitudes of ABRs and vestibular evoked potentials, suggesting synaptic dysfunction in both the auditory and vestibular systems. Whereas ABR thresholds in TSP1 KO mice were relatively unaffected at early ages, TSP1/TSP2 KO mice showed the most severe phenotype among all of the genotypes tested, suggesting functional redundancy between the two genes. On the basis of the above results, we propose that TSPs play an important role in afferent synapse development and function of the inner ear.

Bleomycin-induced lung injury is characterized in the neonatal rat by inflammation dominated by neutrophils and macrophages, inhibited distal airway and vascular development, and pulmonary hypertension, similar to human infants with severe bronchopulmonary dysplasia. Rho-kinase (ROCK) is known to mediate lung injury in adult animals via stimulatory effects on inflammation. We therefore hypothesized that inhibition of ROCK may ameliorate bleomycin-induced lung injury in the neonatal rat. Pups received daily intraperitoneal bleomycin or saline from Postnatal Days 1 through 14 with or without Y-27632, a ROCK inhibitor. Treatment with Y-27632 prevented bleomycin-induced pulmonary hypertension, as evidenced by normalized pulmonary vascular resistance, decreased right-ventricular hypertrophy, and attenuated remodeling of pulmonary resistance arteries. Bleomycin-induced changes in distal lung architecture, including septal thinning, inhibited alveolarization, and decreased numbers of peripheral arteries and capillaries, were partially or completely normalized by Y-27632. Treatment with Y-27632 or a CXCR2 antagonist, SB265610, also abrogated tissue neutrophil influx, while having no effect on macrophages. However, treatment with SB265610 did not prevent bleomycin-induced lung injury. Lung content of angiostatic thrombospondin-1 (TSP1) was increased significantly in the lungs of bleomycin-exposed animals, and was completely attenuated by treatment with Y-27632. Thrombin-stimulated TSP1 production by primary cultured rat pulmonary artery endothelial cells was also attenuated by Y-27632. Taken together, our findings suggest a preventive effect of Y-27632 on bleomycin-mediated injury by a mechanism unrelated to inflammatory cells. Our data suggest that improvements in lung morphology may have been related to indirect stimulatory effects on angiogenesis via down-regulation of TSP1.

BACKGROUND

A disintegrin and metallopeptidase with thrombospondin motif type 1 (ADAMTS1) is a recently discovered metalloproteinase with antiangiogenic activity. The function of ADAMTS1 in gastric cancer remains unknown. Therefore, we were interested in examining ADAMTS1 expression in human gastric cancer, as well as its possible correlation with angiogenesis.

METHODS

The mRNA and protein expression of ADAMTS1, thrombospondin type I (TSP1), and vascular endothelial growth factor (VEGF) was evaluated by RT-PCR and immunohistochemistry, respectively, in 56 paired tumor and normal tissue samples, and corresponding metastatic lymph nodes (n = 42). Microvessel density (MVD) was also evaluated by immunohistochemistry.

RESULTS

ADAMTS1 mRNA and protein levels were significantly lower in primary tumors than in corresponding normal tissues, and were significantly higher in metastatic lymph nodes compared to their matched primary tumors. High ADAMTS1 mRNA and protein expression was found to be significantly associated with lymph node metastasis in primary tumors. There was a negative correlation between ADAMTS1 and VEGF mRNA and protein expression in primary gastric tumors and normal tissues. A negative correlation was also found between ADAMTS1 protein expression and MVD in primary gastric tumors. In contrast, no correlation was detected between ADAMTS1 and TSP1 mRNA and protein expression in primary gastric tumors, normal tissues, and metastatic lymph nodes.

CONCLUSIONS

These findings suggest that ADAMTS1 expression is altered in primary gastric cancer and paired lymph node metastasis. In addition, ADAMTS1 has angioinhibitory effects in primary gastric cancer due to its low expression and negative correlation with VEGF and MVD. However, it appears to lose its anti-angiogenic activity in metastatic lymph nodes in gastric cancer.

Diabetes is a major predictor of in-stent restenosis, which is associated with fibroproliferative remodeling of the vascular wall due to increased transforming growth factor-beta (TGF-beta) action. It is well established that thrombospondin1 (TSP1) is a major regulator of TGF-beta activation in renal and cardiac complications of diabetes. However, the role of the TSP1-TGF-beta pathway in macrovascular diabetic complications, including restenosis, has not been addressed. In mesangial cells, high glucose concentrations depress protein kinase G (PKG) activity, but not PKG-I protein, thereby downregulating transcriptional repression of TSP1. Previously, we showed that high glucose downregulates PKG-I protein expression by vascular smooth muscle cells (VSMCs) through altered NADPH oxidase signaling. In the present study, we investigated whether high glucose regulation of PKG protein and activity in VSMCs similarly regulates TSP1 expression and downstream TGF-beta activity. These studies showed that high glucose stimulates both TSP1 expression and TGF-beta bioactivity in primary murine aortic smooth muscle cells (VSMCs). TSP1 is responsible for the increased TGF-beta bioactivity under high glucose conditions, because treatment with anti-TSP1 antibody, small interfering RNA-TSP1, or an inhibitory peptide blocked glucose-mediated increases in TGF-beta activity and extracellular matrix protein (fibronectin) expression. Overexpression of constitutively active PKG, but not the PKG-I protein, inhibited glucose-induced TSP1 expression and TGF-beta bioactivity, suggesting that PKG protein expression is insufficient to regulate TSP1 expression. Together, these data establish that glucose-mediated downregulation of PKG levels stimulates TSP1 expression and enhances TGF-beta activity and matrix protein expression, which can contribute to vascular remodeling in diabetes.

Enteroaggregative Escherichia coli (EAEC) adherence to human intestinal tissue is known to be mediated by aggregative adherence fimbriae (AAF). However, the host cell molecules involved in EAEC adherence remain uncharacterized. In the present study, four key membrane glycoproteins of cultured human intestinal epithelial cells (INT-407) were found to be involved in the interaction with the T8 strain of EAEC. Nine membrane proteins of INT-407 cells were found to interact with EAEC-T8, of which four were identified as fibronectin, epidermal growth factor receptor (EGFR), Thrombospondin-1 (TSP1) and glucose-regulated protein (GRP-94). Our findings were validated by Western blot using antibody against each identified protein. The adherence of EAEC-T8 to INT-407 cells was reduced to c. 26, 29, 37 and 76% in the presence of the antibody against GRP-94, EGFR, fibronectin and TSP-1, respectively. These findings were further substantiated by flow cytometry, where the final mean fluorescence intensity value of the INT-407 cells (c. 1075) resulting from adherent-labelled bacteria was found to be reduced to c. 26, 132, 228 and 597 in the presence of antibody against GRP-94, EGFR, fibronectin and TSP-1. We propose that GRP-94, EGFR, fibronectin and TSP-1 are involved in the aggregative adherence of EAEC-T8 to INT-407 cells.

To analyze the function of TSP family members, we have expressed and purified mouse TSP1 and TSP3 encoded by recombinant baculoviruses in Spodoptera frugiperda cells and compared these TSPs to mouse TSP2 prepared in a similar way. Yields of purified TSP1 and TSP3 were 5-15 and 2-4 micrograms, respectively, per ml of conditioned medium. Mature, secreted mouse T41 and TSP3 had the previously predicted NH2-terminal sequences of DHVKDTSFDLFSI, and SQDLQVIDLLT, respectively. Analysis by SDS-PAGE and rotary shadowing electron microscopy indicated that TSP1 and TSP2 are disulfide bonded trimers whereas TSP3 is a disulfide-bonded pentamer. Binding assays with 45Ca2+ as ligand and immobilized TSP1, TSP2 and TSP3 demonstrated that all three TSPs are calcium-binding proteins. These results are consistent with previous studies of TSP structure and demonstrate that Spodoptera cells process and secrete TSPs having the same subunit organizations and structure as the native vertebrate molecules.

The extracellular matrix (ECM) acts as a scaffold for kidney cellular organization. Local secretion of the ECM allows kidney cells to readily adapt to changes occurring within the kidney. In addition to providing structural support for cells, the ECM also modulates cell survival, migration, proliferation, and differentiation. Although aberrant regulation of ECM proteins can play a causative role in many diseases, it is not known whether ECM production, cell adhesion, and migration are regulated in a similar manner in kidney epithelial and endothelial cells. Here, we demonstrate that lack of BIM expression differentially impacts kidney endothelial and epithelial cell ECM production, migration, and adhesion, further emphasizing the specialized role of these cell types in kidney function. Bim -/- kidney epithelial cells demonstrated decreased migration, increased adhesion, and sustained expression of osteopontin and thrombospondin-1 (TSP1). In contrast, bim -/- kidney endothelial cells demonstrated increased cell migration, and decreased expression of osteopontin and TSP1. We also observed a fivefold increase in VEGF expression in bim -/- kidney endothelial cells consistent with their increased migration and capillary morphogenesis. These cells also had decreased endothelial nitric oxide synthase activity and nitric oxide bioavailability. Thus kidney endothelial and epithelial cells make unique contributions to the regulation of their ECM composition, with specific impact on adhesive and migratory properties that are essential for their proper function.

The X-ray structure of yeast enolase shows His373 interacting with a water molecule also held by residues Glu168 and Glu211. The water molecule is suggested to participate in the catalytic mechanism (Lebioda, L. and Stec, B. (1991) Biochemistry 30, 2817-2822). Replacement of His373 with asparagine (H373N enolase) or phenylalanine (H373F enolase) reduces enzymatic activity to ca. 10% and 0.0003% of the native enzyme activity, respectively. H373N enolase exhibits a reduced Km for the substrate, 2-phosphoglycerate, and produces the same absorbance changes in the chromophoric substrate analogues TSP1 and AEP1, relative to native enolase. H373F enolase binds AEP less strongly, producing a smaller absorbance change than native enolase, and reacts very little with TSP. H373F enolase dissociates to monomers in the absence of substrate; H373N enolase subunit dissociation is less than H373F enolase but more than native enolase. Substrate and Mg2+ increase subunit association in both mutants. Differential scanning calorimetric experiments indicate that the interaction with substrate that stabilizes enolase to thermal denaturation involves His373. We suggest that the function of His373 in the enolase reaction may involve hydrogen bonding rather than acid/base catalysis, through interaction with the Glu168/Glu211/H2O system, which produces removal or addition of hydroxyl at carbon-3 of the substrate.

OBJECTIVE

Interstitial lung disease accounts for a group of chronic and progressive disorders associated with severe pulmonary vascular remodelling, peripheral vascular rarefaction and fibrosis, thus limiting lung function. We have previously shown that Akt is necessary for myofibroblast differentiation, a critical event in organ fibrosis. However, the contributory role of the Akt-mTOR pathway in interstitial lung disease and the therapeutic benefits of targeting Akt and mTOR remain unclear.

METHODS

We investigated the role of the Akt-mTOR pathway and its downstream molecular mechanisms in chronic hypoxia- and TGFβ-induced pulmonary vascular pruning and fibrosis in mice. We also determined the therapeutic benefits of the Akt inhibitor triciribine and the mTOR inhibitor rapamycin for the treatment of pulmonary fibrosis in mice.

RESULTS

Akt1(-) (/) (-) mice were protected from chronic hypoxia-induced peripheral vascular pruning. In contrast, hyperactivation of Akt1 induced focal fibrosis similar to TGFβ-induced fibrosis. Pharmacological inhibition of Akt, but not the Akt substrate mTOR, inhibited hypoxia- and TGFβ-induced pulmonary vascular rarefaction and fibrosis. Mechanistically, we found that Akt1 modulates pulmonary remodelling via regulation of thrombospondin1 (TSP1) expression. Hypoxic Akt1(-) (/) (-) mice lungs expressed less TSP1. Moreover, TSP1(-) (/) (-) mice were resistant to adMyrAkt1-induced pulmonary fibrosis.

CONCLUSIONS

Our study identified Akt1 as a novel target for the treatment of interstitial lung disease and provides preclinical data on the potential benefits of the Akt inhibitor triciribine for the treatment of interstitial lung disease.

Integration of stem cell-derived neurons into the central nervous system (CNS) remains a challenge. A co-culture system is developed to understand whether mouse embryonic stem cell (ESC)-derived spiral ganglion neuron (SGN)-like cells (ESNs) synapse with native mouse cochlear nucleus (CN) neurons. A Cre system is used to generate Cop-GFP ESCs, which are induced into ESNs expressing features similar to auditory SGNs. Neural connections are observed between ESNs and CN neurons 4-6 days after co-culturing, which is stimulated by thrombospondin-1 (TSP1). Antagonist and loss-of-function small hairpin RNA studies indicate that the α2δ1 receptor is critical for TSP1-induced synaptogenesis effects. Newly generated synapse-like structures express pre- and post-synaptic proteins. Synaptic vesicle recycling, pair recording, and blocker electrophysiology suggest functional synaptic vesicles, transsynaptic activities, and formation of glutamatergic synapses. These results demonstrate the synaptogenesis capability of ESNs, which is important for pluripotent ESC-derived neurons to form functional synaptic connections to CNS neurons.

In this study, we examined the binding of soluble TSP1 (and ox-LDL) to CD36-transfected cells and the mechanisms by which immobilized TSP1 mediated attachment and haptotaxis (cell migration towards a substratum-bound ligand) of these transfected cells. CD36 cDNA transfection of NIH 3T3 cells clearly induced a dramatic increase in binding of both soluble [125I]-TSP1 and [125I]-ox-LDL to the surface of CD36-transfected cells, indicating that there was a gain of function with CD36 transfection in NIH 3T3 cells. Despite this gain of function, mock- and CD36-transfected NIH 3T3 cells attached and migrated to a similar extent on immobilized TSP1. An anti-TSP1 oligoclonal antibody inhibited CD36-transfected cell attachment to TSP1 while function blocking anti-CD36 antibodies, alone or in combination with heparin, did not. A series of fusion proteins encompassing cell-recognition domains of TSP1 was then used to delineate mechanisms by which NIH 3T3 cells adhere to TSP1. Although CD36 binds soluble TSP1 through a CSVTCG sequence located within type 1 repeats, 18,19CD36-transfected NIH 3T3 cells did not attach to immobilized type 1 repeats while they did adhere to the N-terminal, type 3 repeats (in an RGD-dependent manner) and the C-terminal domain of TSP1. Conversely, Bowes melanoma cells attached to type 1 repeats and the N- and C-terminal domains of TSP1. However, CD36cDNA transfection of Bowes cells did not increase cell attachment to type 1 repeats compared to that observed with mock-transfected Bowes cells. Moreover, a function blocking anti-CSVTCG peptide antibody did not inhibit the attachment of mock- and CD36-transfected Bowes cells to type 1 repeats. It is suggested that CD36/TSP1 interaction does not occur upon cell-matrix adhesion and haptotaxis because TSP1 undergoes conformational changes that do not allow the exposure of the CD36 binding site.

OBJECTIVE

Lichen sclerosus (LS) is a chronic inflammatory disease of the genital skin of unknown aetiology. The role of LS in penile squamous cell carcinogenesis is not well characterized. HPV has been implicated in both, as have epigenetic changes. The presence of HPV and hypermethylation of the MGMT, p16, RASSF1, RASSF2, TSLC1 and TSP1 genes were studied in penile LS; MGMT, RASSF2 and TSLC1 hypermethylation in penile cancer and TSLC1 hypermethylation in vulvar LS and cancer extends previous results reported by our group.

RESULTS

Thirty-seven HPV genotypes and hypermethylation were evaluated by PCR/reverse-line-blot and methylation-specific PCR respectively, in 27 preputial LS, 24 penile SCC, 30 vulvar SCC, 21 vulvar LS and 22 normal skin cases. HPV66 was present in 3.7% of penile LS cases, and p16 and RASSF2 hypermethylation were more frequent in penile cancer than in penile LS. p16, RASSF1, RASSF2 and TSP1 hypermethylation were similar in penile and vulvar LS.

CONCLUSIONS

Gene hypermethylation is a common event in penile LS, and occurs approximately as frequently as in vulvar LS. Certain genes can be hypermethylated as an early or late event in LS or cancer, respectively. This suggests a possible sequential role for these alterations in the transition from benign to malignant lesions.