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PURPOSE: Orally administered paclitaxel offers increased patient convenience while providing a method to prolong exposure without long continuous, or repeated, intravenous infusions. The oral bioavailability of paclitaxel is improved through co-administration with ritonavir and application of a suitable pharmaceutical formulation, which addresses the dissolution-limited absorption of paclitaxel. We aimed to characterize the pharmacokinetics of different paclitaxel formulations, co-administered with ritonavir, and to investigate a pharmacodynamic relationship between low-dose metronomic (LDM) treatment with oral paclitaxel and the anti-angiogenic marker thrombospondin-1 (TSP-1). METHODS: Fifty-eight patients treated with different oral paclitaxel formulations were included for pharmacokinetic analysis. Pharmacodynamic data was available for 36 patients. All population pharmacokinetic/pharmacodynamic modelling was performed using non-linear mixed-effects modelling. RESULTS: A pharmacokinetic model consisting of gut, liver, central, and peripheral compartments was developed for paclitaxel. The gastrointestinal absorption rate was modelled with a Weibull function. Relative gut bioavailabilities of the tablet and capsule formulations, as fractions of the gut bioavailability of the drinking solution, were estimated to be 0.97 (95%CI: 0.67-1.33) and 0.46 (95%CI: 0.34-0.61), respectively. The pharmacokinetic/pharmacodynamic relationship between paclitaxel and TSP-1 was modelled using a turnover model with paclitaxel plasma concentrations driving an increase in TSP-1 formation rate following an Emax relationship with an EC50 of 284 ng/mL (95%CI: 122-724). CONCLUSION: The developed pharmacokinetic model adequately described the paclitaxel plasma concentrations for the different oral formulations co-administered with ritonavir. This model, and the established pharmacokinetic/pharmacodynamic relationship with TSP-1, may facilitate future development of oral paclitaxel.

Purpose: Conjunctival squamous cell carcinoma (SCC) is primarily treated with surgical resection. SCC has various stages, and local recurrence is common. The purpose of this study was to investigate thrombospondin-1 expression and its association with prognosis. ...

Background: Thrombospondin-1 (TSP-1), a Ca2+-binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions. Signaling involving TSP-1, through its cognate receptor CD47, orchestrates a wide array of cellular functions including cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis. In the present study, we investigated the impact of TSP-1/CD47 signaling on Ca2+ dynamics, survival, and deformability of human red blood cells (RBCs). Methods: Whole-cell patch-clamp was employed to examine transmembrane cation conductance. RBC intracellular Ca2+ levels and multiple indices of RBC cell death were determined using cytofluorometry analysis. RBC morphology and microvesiculation were examined using imaging flow cytometry. RBC deformability was measured using laser-assisted optical rotational cell analyzer. Results: Exposure of RBCs to recombinant human TSP-1 significantly increased RBC intracellular Ca2+ levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca2+ influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca2+- and nitric oxide-mediated RBC cell death. Monoclonal (mouse IgG1) antibody-mediated CD47 ligation using 1F7 recapitulated the cell death-inducing effects of TSP-1. Furthermore, TSP-1 treatment altered RBC cell shape and stiffness (maximum elongation index). Conclusions: Taken together, our data unravel a new role for TSP-1/CD47 signaling in mediating Ca2+ influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases.

BACKGROUND. Neoadjuvant hormonal therapy (NHT) is performed to improve the outcome in organ-confined prostate cancer. However, there is little information regarding the relationship between angiogenesis and NHT. The aim of this study was to identify a suitable method to evaluate the angiogenic status of tissue, and to determine the prognostic value of this method for biochemical recurrence in patients who had undergone radical prostatectomy after NHT. METHODS. We analyzed 108 formalin-fixed specimens from patients treated by radical prostatectomy. NHT was administered in 48 patients (52.9%) and 60 patients who had a similar Gleason score and pT stage were selected as a non-NHT treated control group. Microvessel density (MVD) was measured using anti-CD31, anti-CD34, and anti-CD105 antibodies. The expressions of vascular endothelial growth factor (VEGF)-A and thrombospondin (TSP)-1 were also evaluated by immunohistochemistry. The prognostic value of CD31-, CD34-, and CD105-MVD for biochemical recurrence was investigated. RESULTS. The mean/SD of CD105-MVD in the NHT group (13.3/4.7) was significantly (P<0.001) lower than that in the non-NHT group (125.8/7.3). In the NHT group, CD105-MVD was associated with pT stage and it was positively correlated with VEGF-A expression (r=0.56, P<0.001) and negatively correlated with TSP-1 expression (r=0.42, P=0.003). CD105-MVD was identified as a significant predictor of biochemical recurrence (BCR) in patients treated with NHT (log rank test, P<0.001). Although CD31- and CD34-MVD were significantly associated with pT stage or Gleason score in non-NHT group, they were not associated with pathological features and BCR in NHT group. CONCLUSIONS. Our results indicate that CD105-MVD reflects the angiogenic conditions in prostate cancer tissues treated with NHT. CD105-MVD was also identified as a significant and independent predictor of biochemical recurrence in prostate cancer patients who underwent radical prostatectomy with NHT., The Prostate, 75(1), pp.84-91; 2015

Background: The pathogenesis of idiopathic pulmonary fibrosis (IPF) in dogs is poorly understood. In human, transforming growth factor β1 (TGF-β1) is considered central in the pathogenesis. Objectives: To investigate TGF-β1 pathway in IPF. Animals: Lung tissues from 12 affected and 11 control dogs. Serum from 16 affected West Highland white Terriers (WHWTs) and healthy dogs from predisposed (13 WHWTs, 12 Scottish Terriers and 13 Bichons Frise) and nonpredisposed breeds (10 Whippets, 10 Belgian shepherds, 8 Labradors). Methods: In this prospective study, immunohistochemistry was used to evaluate expression and localization of TGF-β1 protein and proteins involved in TGF-β1 signaling (TGF-β receptor type I and phospho-Smad2/3). Pulmonary expression of TGF-β1 and molecules involved in its storage (latent TGF-β binding proteins [LTBP] 1, 2, and 4), activation (ανβ6 and ανβ8 integrins, thrombospondin-1) and signal inhibition (Smad 7) was analyzed by quantitative reverse transcriptase PCR. Circulating TGF-β1 concentration was measured by ELISA. Results: In IPF, high level of TGF-β1 protein was found in areas of fibrosis, epithelial cells had strong expression of TGF-β receptor type 1 and phospho-Smad2/3, gene expression was decreased for LTBP 4 (P =.009) and β8 integrin (P <.001) and increased for thrombospondin-1 (P =.016); no difference was seen for Smad7, LTBP1 and 2. Serum TGF-β1 concentration was higher in predisposed compared with nonpredisposed breeds (P <.0001). Conclusions and Clinical Importance: This study identified an enhanced TGF-β1 signaling activity in IPF. TGF-β1 storage and activation proteins with altered expression represent potential therapeutic targets. Higher circulating TGF-β1 concentration in predisposed breeds might partly explain their susceptibility for IPF., SCOPUS: ar.j, info:eu-repo/semantics/published

Thrombospondin-1 (TSP-1) binding to its membrane receptor CD47 results in an inhibtion of the nitric oxide (NO) receptor soluble guanylate cyclase (sGC) and a decrease in intracellular cGMP levels. This causes physiologic effects such as vasoconstriction and a rise in blood pressure. The mechanism by which TSP-1 binds to CD47 at the membrane to decrease sGC activity is largely unknown. CD47 can physically associate with a number of binding partners, including α(v)β₃ and vascular endothelial growth factor receptor 2 (VEGFR2). Binding of a C-terminal fragment of TSP-1 called E3CaG1 to CD47 leads to a rise in intracellular calcium ([Ca²⁺](i)), which decreases sGC activity via a phosphorylation event. Binding of E3CaG1 is also known to disrupt the interaction between CD47 and VEGFR2, leading to a decrease in endothelial nitric-oxide synthase (eNOS) activity and cGMP levels through an Akt signaling pathway. However, it is not known whether other membrane proteins associated with CD47 are required for E3CaG1 binding and a subsequent [Ca²⁺](i) increase. Plasmon-waveguide resonance (PWR) spectroscopy was employed to elucidate the mechanism of TSP-1 inhibition of sGC activity through membrane complexes involving CD47. Using PWR, I found E3CaG1 can bind specifically to CD47 within native Jurkat membranes with picomolar and nanomolar dissociation constants (K(d)), suggesting multiple CD47 complexes are present. Among these complexes, CD47/VEGFR2 was found to bind E3CaG1 with a picomolar K(d)and CD47/α(v)β₃ was found to bind E3CaG1 with a nanomolar K(d). In addition, the presence of an anti-VEGFR2 antibody inhibited the E3CaG1-induced calcium response, which suggested CD47 in complex with VEGFR2 was responsible for TSP-1 reduction of sGC activity. I show that when both CD47 and VEGFR2 are returned to a HEK 293T cell line that does not contain these receptors, an increase in [Ca²⁺](i) upon E3CaG1 binding is restored. Interestingly, E3CaG1 was also found to bind to VEGFR2 in comp

Desmoplastic (scirrhous) invasion and lymph node metastasis are critical for the treatment and prognosis of invasive ductal carcinoma of the breast. Despite being an anti-angiogenic therapeutic candidate, Thrombospondin-1 (TSP-1) promotes invasion and metastasis of some carcinomas. To clarify the effect of TSP-1 on invasion and metastasis, we obtained 101 invasive ductal carcinomas of the breast with axillary lymph node resection. All tumors were histologically divided into two categories, carcinomas with, and those with non- /minimal desmoplastic component. Immunohistochemistry for TSP-1 was performed on all primary tumors and axillary lymph nodes with tumor metastasis. Fifty-four (53.5%) of 101 tumors were recognized as positive for TSP-1 in the cytoplasm of tumor cells. Histological study showed that significantly more cancers with desmoplastic components (46/69, 66.7%) manifested TSP-1 expression than did cancers with no- or minimal (less than 20%) desmoplasia (8/32, 25.0% ; p< 0.001). Axillary lymph node metastasis was significantly higher in TSP-1-positive- (28/54, 51.9%) than TSP-1-negative cancers (11/47, 23.4% ; p<0.005). The present study indicates that tumor cells in the desmoplastic component strongly expressed TSP-1 in invasive ductal carcinoma of the breast and TSP-1 participates in invasion of these tumors. Our findings also suggest that TSP-1 promotes lymph node metastasis and TSP-1 potentially could be a predictive marker for metastasis.

Leung, Winnie., Thesis (M.Phil.)--Chinese University of Hong Kong, 2011., Includes bibliographical references (leaves 155-164)., s in English and Chinese., p.i, 摘要 --- p.iii, Acknowledgements --- p.v, Table of Contents --- p.vi, List of Figures --- p.x, Abbreviations --- p.xii, Chapter Chapter 1 --- General Introduction --- p.1, Chapter 1.1 --- Clinical management of Cancer --- p.2, Chapter 1.2 --- Multidrug resistance --- p.8, Chapter 1.3 --- Aim of study --- p.14, Chapter Chapter 2 --- Identification of gene contributing to multidrug resistance in human uterine sarcoma cells --- p.16, Chapter 2.1 --- Introduction --- p.17, Chapter 2.2 --- Material and Methods, Chapter 2.2.1 --- Materials, Chapter 2.2.1.1 --- Cell lines --- p.20, Chapter 2.2.1.2 --- "Cell culture medium, supplements and buffers" --- p.20, Chapter 2.2.1.3 --- Gene expression assay reagents --- p.22, Chapter 2.2.1.4 --- Western blotting reagents --- p.24, Chapter 2.2.1.5 --- MTT assay reagents --- p.29, Chapter 2.2.1.6 --- Apoptosis analysis by flow cytometry reagents --- p.29, Chapter 2.2.2 --- Metho --- p.ds, Chapter 2.2.2.1 --- Cell Culture --- p.31, Chapter 2.2.2.2 --- MTT assay --- p.32, Chapter 2.2.2.3 --- Gene expression essay (RT-PCR) --- p.33, Chapter 2.2.2.4 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein lysate and Western blotting --- p.37, Chapter 2.2.2.5 --- Quantification of doxorubicin uptake by flow cytometry --- p.40, Chapter 2.2.2.6 --- Apoptosis analysis by flow cytometry --- p.41, Chapter 2.3 --- Results --- p.4, Chapter 2.3.1 --- Cytotoxicity of doxorubicin on SA and DX5 cells --- p.43, Chapter 2.3.2 --- mRNA expression of multidrug resistance related genes in SA and DX5 cells --- p.46, Chapter 2.3.3 --- P-glycoprotein expression in SA and DX5 cells --- p.49, Chapter 2.3.4 --- Doxorubicin (Dox) uptake by SA and DX5 cells --- p.51, Chapter 2.3.5 --- Doxorubicin induced Apoptosis in SA and DX5 cells --- p.54, Chapter 2.4 --- Discussion --- p.61, Chapter 2.5 --- Conclusion --- p.65, Chapter Chapter 3 --- Alternation in P-glycoprotein expression in DX5_Ninjl cells --- p.66, Chapter 3.1 --- Introduction --- p.67, Chapter 3.2 --- Materials and Methods, Chapter 3.2.1 --- Materials, Chapter 3.2.1.1 --- Cell lines --- p.70, Chapter 3.2.1.2 --- "Cell culture medium, supplements and buffers" --- p.70, Chapter 3.2.1.3 --- Gene expression assay reagents --- p.70, Chapter 3.2.1.4 --- Western blotting reagents --- p.72, Chapter 3.2.1.5 --- Plasmid DNA extraction --- p.75, Chapter 3.2.1.6 --- Transient transfection --- p.76, Chapter 3.2.1.7 --- MTT reagents --- p.76, Chapter 3.2.2 --- Methods, Chapter 3.2.2.1 --- Cell culture --- p.78, Chapter 3.2.2.2 --- Gene expression essay (RT-PCR) --- p.79, Chapter 3.2.2.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein lysate and Western blotting --- p.81, Chapter 3.2.2.4 --- DNA plasmid extraction --- p.83, Chapter 3.2.2.5 --- Transient transfection --- p.84, Chapter 3.2.2.6 --- MTT assay --- p.85, Chapter 3.2.2.7 --- Quantification of doxorubicin (Dox) uptake by flow cytometry --- p.86, Chapter 3.3 --- Results, Chapter 3.3.1 --- mRNA expression of Ninjurinl (Ninj1) in SA and DX5 cells --- p.87, Chapter 3.3.2 --- The protein expression of Ninjurinl (Ninj1) in SA and DX5 cells --- p.89, Chapter 3.3.3 --- Ninjurin1 (Ninj1) cDNA transfection in DX5 cells --- p.91, Chapter 3.3.4 --- mRNA expression of MDR1 in Ninjurin1-transfected DX5 cells (DX5_Ninjl) --- p.93, Chapter 3.3.5 --- P-glycoprotein expression in Ninjurin1-transfected DX5 cells --- p.95, Chapter 3.3.6 --- "Cytotoxicity of doxorubicin (Dox) on DX5 control, DX5 vector control and DX5_Ninjl cells" --- p.97, Chapter 3.3.7 --- "Doxorubicin (Dox) uptake by SA control, DX5 control and DX5_Ninjl cells" --- p.99, Chapter 3.4 --- Discussion --- p.102, Chapter 3.5 --- Conclusion --- p.105, Chapter Chapter 4 --- Alternation in MDR1 expression in DX5一THBS1 cells --- p.106, Chapter 4.1 --- Introduction --- p.107, Chapter 4.2 --- Materials and Methods, Chapter 4.2.1 --- Materials, Chapter 4.2.1.1 --- Cell lines --- p.109, Chapter 4.2.1.2 --- Cell culture medium； supplements and buffers --- p.109, Chapter 4.2.1.3 --- Gene expression assay reagents --- p.109, Chapter 4.2.1.4 --- Western blotting reagents --- p.111, Chapter 4.2.1.5 --- Plasmid DNA extraction --- p.114, Chapter 4.2.1.6 --- Transient transfection --- p.115, Chapter 4.2.1.7 --- MTT reagents --- p.115, Chapter 4.2.2 --- Methods, Chapter 4.2.2.1 --- Cell culture --- p.117, Chapter 4.2.2.2 --- Gene expression essay (RT-PCR) --- p.118, Chapter 4.2.2.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein lysate and Western blotting --- p.120, Chapter 4.2.2.4 --- DNA plasmid extraction --- p.123, Chapter 4.2.2.5 --- Transient transfection --- p.123, Chapter 4.2.2.6 --- MTT assay --- p.124, Chapter 4.2.2.7 --- Quantification of doxorubicin (Dox) uptake by flow cytometry --- p.125, Chapter 4.3 --- Results, Chapter 4.3.1 --- mRNA expression of Thrombospondinl (THBS1) in SA and DX5 cells --- p.126, Chapter 4.3.2 --- The protein expression of Thrombospondinl (THBS1) in SA and DX5 cells --- p.128, Chapter 4.3.3 --- Thrombospondinl (THBS1) cDNA transfection in DX5 cells --- p.130, Chapter 4.3.4 --- mRNA expression of MDR1 in Thrombospondinl-transfected DX5 cells (DX5_THBS1) --- p.132, Chapter 4.3.5 --- P-glycoprotein expression in Thrombospondinl-transfected DX5 cells --- p.134, Chapter 4.3.6 --- "Cytotoxicity of doxorubicin (Dox) on DX5 control, DX5 vector control and DX5一THBS1 cells" --- p.136, Chapter 4.3.7 --- "Doxorubicin (Dox) uptake by SA control, DX5 control and DX5_THBS1 cells" --- p.138, Chapter 4.4 --- Discussion --- p.141, Chapter 4.5 --- Conclusion --- p.145, Chapter Chapter 5 --- General discussion --- p.146, Chapter 5.1 --- Doxorubicin induced multidrug resistance in human uterin sarcoma cells via upregulation of P-glycoprotein --- p.147, Chapter 5.2 --- The down-regulation of Ninjurin1 in human uterine sarcoma cells contributed to multidrug resistance --- p.148, Chapter 5.3 --- The down-regulation of Thrombospondin1 in human uterine sarcoma cells contributed to multidrug resistance --- p.150, Chapter 5.4 --- Conclusion and Future Perspective --- p.153, Reference --- p.155, http://library.cuhk.edu.hk/record=b5894755, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Ng, Ka Yan., Thesis (M.Phil.)--Chinese University of Hong Kong, 2010., Includes bibliographical references (leaves 118-131)., s in English and Chinese., Thesis Abstract --- p.i, 論文摘要 --- p.vi, Acknowledgements --- p.x, Abbreviations --- p.xii, Thesis Content --- p.xv, List of Figures --- p.xix, List of Tables --- p.xxi, Chapter Chapter 1 --- Introduction --- p.1, Chapter 1.1 --- Haematopoiesis --- p.1, Chapter 1.2 --- Leukemia --- p.2, Chapter 1.3 --- Childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) --- p.3, Chapter 1.3.1 --- Epidemiology --- p.4, Chapter 1.3.2 --- Causes and risk factors --- p.4, Chapter 1.3.3 --- Clinical features --- p.6, Chapter 1.3.4 --- Morphology --- p.6, Chapter 1.4 --- Classification of BCP-ALL --- p.7, Chapter 1.4.1 --- Immunophenotyping --- p.7, Chapter 1.4.2 --- Cytogenetics and molecular genetics --- p.9, Chapter 1.5 --- Prognostic factors --- p.13, Chapter 1.6 --- Current treatments of BCP-ALL --- p.15, Chapter Chapter 2 --- Literature Review --- p.18, Chapter 2.1 --- Cytogenetics abnormalities in BCP-ALL --- p.18, Chapter 2.1.1 --- Chromosomal translocation --- p.18, Chapter 2.1.2 --- Aneuploidy --- p.21, Chapter 2.2 --- Epigenetic aberrations --- p.21, Chapter 2.2.1 --- DNA methylation --- p.22, Chapter 2.2.2 --- Mechanism of DNA Methylation in Transcription Repression --- p.23, Chapter 2.3 --- DNA Methylation in Normal Haematopoiesis --- p.25, Chapter 2.4 --- DNA Methylation in Haematological Malignancies --- p.26, Chapter 2.4.1 --- DNA methylation in ALL --- p.26, Chapter 2.4.2 --- DNA methylation in BCP-ALL --- p.29, Chapter 2.5 --- Angiogenesis in pathogenesis of acute leukemias --- p.30, Chapter 2.6 --- Thrombospondin-1 (TSP-1) --- p.32, Chapter 2.6.1 --- Structure of TSP-1 --- p.33, Chapter 2.6.2 --- The role of TSP-1 in tumorigenesis --- p.34, Chapter 2.6.3 --- TSP-1 mediates the activation of TGFβ --- p.36, Chapter 2.6.4 --- TSP-1 mediates TGFβ-induced Apoptosis --- p.37, Chapter 2.6.5 --- Association of TGFβ with normal haematopoiesis and haematological malignancies progression --- p.40, Chapter 2.6.6 --- TSP-1 Induced Apoptosis via its Receptor CD36 --- p.42, Chapter 2.6.7 --- THBS1 promoter hypermethylation and its association with tumorigenesis --- p.43, Chapter 2.6.8 --- Effect of THBS1 aberrant methylation on TGFp --- p.45, Chapter Chapter 3 --- Rationale of Study --- p.47, Chapter Chapter 4 --- Materials and Methods --- p.52, Chapter 4.1 --- Patient sample --- p.52, Chapter 4.2 --- Cell lines --- p.52, Chapter 4.3 --- Mononuclear cells isolation --- p.53, Chapter 4.4 --- THBS1 promoter hypermethylation analysis --- p.54, Chapter 4.4.1 --- DNA extraction from mononuclear cells and cell lines --- p.54, Chapter 4.4.2 --- Bisulfite conversion --- p.55, Chapter 4.4.3 --- Methylation specific PCR (MSP) --- p.55, Chapter 4.5 --- Quantification of THBS1 mRNA expression --- p.57, Chapter 4.5.1 --- RNA extraction --- p.57, Chapter 4.5.2 --- Reverse transcription PCR --- p.58, Chapter 4.5.3 --- Real-time RT-PCR --- p.58, Chapter 4.6 --- Determination of plasma TSP-1 level --- p.59, Chapter 4.7 --- TSP-1 treatment --- p.60, Chapter 4.8 --- Flow cytometry analysis --- p.60, Chapter 4.8.1 --- Annexin-V analysis --- p.60, Chapter 4.8.2 --- Cell fixation --- p.61, Chapter 4.8.3 --- Analysis of Caspase-3 activation --- p.62, Chapter 4.8.4 --- "Analysis of TGFβ downstream pathway activation: Phosphorylation of Smad2/3, JNK and p38" --- p.62, Chapter 4.9 --- Determination ofTGF-β expression --- p.63, Chapter 4.10 --- Statistical analysis methods --- p.64, Chapter Chapter 5 --- Results, Chapter 5.1 --- THBS1 methylation statuses in BCP-ALL patients and cell lines --- p.66, Chapter 5.2 --- Correlation of THBS1 methylation statuses and clinico- pathological features in BCP-ALL patients --- p.68, Chapter 5.3 --- Association of THBS1 methylation and THBS1 mRNA expression --- p.69, Chapter 5.4 --- Effect of TSP-1 treatment on apoptosis level of BCP-ALL cells --- p.72, Chapter 5.4.1 --- Annexin-V assay --- p.72, Chapter 5.4.2 --- Caspase-3 activation assay --- p.75, Chapter 5.5 --- THBS1 methylation and activation of secreted TGFβ --- p.78, Chapter 5.6 --- Effect of TSP-1 treatment on activation of TGFβ --- p.80, Chapter 5.7 --- The involvement ofTGFβ activation in TSP-1 induced apoptosis in BCP-ALL --- p.82, Chapter 5.8 --- The association of TGFβ signaling pathway activities with THBS1 methylation --- p.86, Chapter Chapter 6 --- Discussion --- p.91, Chapter 6.1 --- THBS-1 promoter hypermethylation in BCP-ALL cell lines and patients: Correlation with expression and clinico-pathological profile --- p.93, Chapter 6.1.1 --- THBS1 promoter hypermethylation status in childhood BCP-ALL --- p.93, Chapter 6.1.2 --- THBS1 methylation as prognostic markers --- p.94, Chapter 6.1.3 --- "Association of THBS1 methylation status, mRNA expression and TSP-1 protein expression in childhood BCP-ALL" --- p.96, Chapter 6.2 --- Study of the correlation of TSP-1 induced apoptosis with the THBS1 promoter methylation status --- p.99, Chapter 6.3 --- Elucidation of the molecular mechanisms of TSP-1 induced apoptosis: study of the involvement of TGFβ activation --- p.103, Chapter 6.3.1 --- Latent TGFβ activation by TSP-1 in BCP-ALL and association with THBS1 methylation status --- p.103, Chapter 6.3.2 --- TSP-1 induced cell death through activation ofTGFβ --- p.105, Chapter 6.3.3 --- TSP-1 induced apoptotic signals via TGFβ signaling pathway --- p.107, Chapter 6.4 --- Limitation of study --- p.113, Chapter 6.5 --- Future studies --- p.114, Chapter 6.5.1 --- Continuation study in TSP-1 induced TGFβ-mediated pathways --- p.114, Chapter 6.5.2 --- Microarray analysis --- p.115, Chapter 6.6 --- TSP-1 in treatment of childhood BCP-ALL --- p.115, Chapter Chapter 7 --- Conclusion --- p.117, Reference --- p.118, http://library.cuhk.edu.hk/record=b5894335, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

BACKGROUND: Angiogenesis is the formation of new blood vessels by capillary sprouting from pre-existing vessels. Tumor growth is angiogenesis-dependent and the formation of new blood vessels is associated with the increased expression of angiogenic factors. Prostasomes are secretory granules produced, stored and released by the glandular epithelial cells of the prostate. We investigated the expression of selected angiogenic and anti-angiogenic factors on the surface of prostasomes of different origins as well as the direct effect of prostasomes on angiogenesis. METHODS: VEGF, endothelin-1, endostatin, and thrombospondin-1 were determined on prostasomes from seminal fluid and human prostate cancer cell lines (DU145,PC-3,LNCaP) using different immunochemical techniques. Human dermal microvascular endothelial cells were incubated with seminal and DU145 cell-prostasomes and with radioactive thymidine. The effect of prostasomes on angiogenesis was judged by measuring the uptake of labeled thymidine. The presence of any deleterious effects of prostasomes on the endothelial cells was investigated using thymidine assay and confocal laser microscopy. RESULTS: VEGF and endothelin-1 were determined on malignant cell-prostasomes (no difference between cell lines) but not determined on seminal prostasomes. The same applies for the expression of endostatin but with much higher expression on malignant cell-prostasomes with obvious differences between them. Seminal and DU145 cell-prostasomes were found to have anti-angiogenic effect which was more expressed by DU145 cell-prostasomes. No deleterious effect of prostasomes on endothelial function was detected using either thymidine assay or microscopy. CONCLUSIONS: Prostasomes contain pro- and anti-angiogenic factors that function to counteract each other unless the impact from one side exceeds the other to bring about dysequilibrium.

In the present work the time sequence of blood activation by alumina membranes with different porosities (20 and 200 nm in diameter) was studied. The membranes were incubated with whole blood from 2 min to 4 h. Platelet adhesion and activation in addition to complement activation were monitored at different time points. Evaluation of platelet adhesion and activation was done by determining the change in platelet number and the levels of thrombospondin-1 in the fluid phase. Scanning electron microscopy studies were done to further evaluate platelet adhesion and morphology. Immunocytochemical staining was used to evaluate the presence of CD41 and CD62P antigens on the material surface. Complement activation was monitored by measuring C3a and sC5b-9 in plasma samples by means of enzyme immunoassays. Both alumina membranes displayed similar complement activation time profiles, with levels of C3a and sC5b-9 increasing with incubation time. A statistically significant difference between the membranes was found after 60 min of incubation. Platelet activation characteristics and time profile were different between the two membranes. Platelet adhesion increased over time for the 20 nm surface, while the clusters of microparticles on the 200 nm surface did not appreciably change during the course of the experiment. The release of thrombospondin-1 increased with time for both membranes, however much later for the 200 nm alumina (240 min) as compared to the 20 nm membrane (60 min). The surface topography of the alumina most probably influence protein transition rate, which in turn affects material-platelet activation kinetics.

Ischaemia/reperfusion (IR) is the commonest cause of acute kidney injury which is a major cause of morbidity and an important contributing factor to mortality in patients with a wide range of medical and surgical conditions. IR often occurs in an unpredictable manner but in certain situations, most notably organ transplantation its occurrence can be pre-empted thus allowing consideration of preventative therapies. IR injury is an inflammatory process involving complex interactions between resident and infiltrating cells of both innate and cognate arms of the immune system. The immediate sequelae of renal IR are necrosis of tubular epithelial cells associated with an acute decline in renal function. Chronic kidney injury may follow and is preceded by endothelial cell injury and loss of peritubular capillaries. The mechanisms linking acute and chronic injury, or more specifically acute inflammatory tubular damage and long-term endothelial cell injury, are not well understood. The tissue factor (TF) initiated coagulation pathway has been shown to contribute to renal IR injury. Signalling by TF and the coagulation protease thrombin leads to chemokine upregulation, neutrophil infiltration and kidney injury after IR. These effects are primarily dependent on activation of the thrombin receptor protease activated receptor (PAR)-1. TF and thrombin may promote survival and growth of endothelial cells; therefore it is possible that inhibition of TF or PAR-1 signalling could have deleterious effects on endothelial cells after IR. The primary aims of the studies described in this thesis were two-fold:-1. To characterise the expression of factors contributing to endothelial cell survival or apoptosis after renal IR and determine whether the TF-thrombin-PAR-1 system is involved in their regulation.2. To determine whether signalling by the cytoplasmic domain of TF, which does not promote thrombin production but may signal directly, is involved in renal IR injury. ResultsA murine mo

Transplantation of pancreatic islets is clinically used to treat type 1 diabetes, but requires multiple donors. Previous experimental studies have demonstrated that transplanted islets have a low blood vessel density, which leads to a hypoxic microenvironment. The present study tested the hypothesis that experimental prolactin pretreatment, a substance which seems to stimulate angiogenesis in endogenous islets, would increase graft blood vessel density, thereby improving transplantation outcome. Pancreatic islets from C57BL/6 mice were incubated with prolactin (500 ng/ml) or vehicle during the last 24 h of culture before syngeneic transplantation beneath the renal capsule, or recipients were injected with prolactin or vehicle for the first 7 days after transplantation. One month post-transplantation, graft vascular density, blood flow, oxygen tension, endocrine volume and function was evaluated. Also human islets were incubated with prolactin or vehicle before experimental transplantation and investigated for vascular engraftment. Vascular engraftment of syngeneically transplanted mouse islets was improved by both in vivo and in vitro prolactin pretreatment. Moreover, prolactin pretreatment in vitro of islets used for transplantation improved recovery from diabetes in a minimal islet mass model. Interestingly, also human islets subjected to prolactin treatment before experimental transplantation demonstrated improved revascularization, blood perfusion and oxygen tension when evaluated one month post-transplantation. We conclude that prolactin may improve engraftment of transplanted pancreatic islets. The protocol with pretreatment of islets ex vivo could minimize the risk of side effects when used in the clinical setting.

New blood vessel formation in transplanted islets occurs within 7-14 days posttransplantation through both the expansion of donor islet endothelium and ingrowth of blood vessels from the implantation organ. However, several studies indicate that although the islets attract recipient blood vessels, the formed intra-islet vascular network is insufficient, which affects islet posttransplant function. This study aimed to develop an in vitro model to investigate the migration and proliferation properties of isolated liver and islet endothelium. Rat islet or liver endothelium was purified using Bandeiraea simplicifolia (BS-1)-coated Dynabeads. The liver endothelium displayed an increased migration towards islet-conditioned medium, and this chemo-attractant effect was fully prevented by adding a neutralizing vascular endothelial growth factor (VEGF)-antibody. In contrast, islet-produced VEGF failed to induce islet endothelial cell migration and only had marginal effects on islet endothelial cell proliferation. These properties could, however, be activated through blocking the effects of either endostatin, thrombospondin-1 or α1-antitrypsin. In conclusion, VEGF may attract recipient blood vessels towards intrahepatically transplanted islets, but intra-islet vascular expansion is hampered by angiostatic factors present within the islets and the islet endothelium. Inhibition of these early after transplantation may provide a strategy to restore the islet vascular network and improve islet graft function.

Our overall goal is to determine if human breast cancer can be prevented from becoming angiogenic when it is still at a microscopic size (< ~ 1 mm3). Some of our key research accomplishments this year are: (i) Heat shock protein-27 (HSP-27) was identified as a gene that was upregulated 27-fold in the angiogenic breast cancer cells MDA-MB-436 as compared to the non-angiogenic dormant breast cancer cells. (ii) Increased expression of HSP-27 was validated by Western blot analysis. (iii) Confirmation of decreased expression of the endogenous angiogenesis inhibitors thrombospondin-1 and endostatin in breast cancer cell lines with a BRCA1 mutation (HCC937). (iv) Demonstration by immunofluorescence of decreased thrombospondin-1 and endostatin expression in sections from human breast tumors with BRCA1 mutations as compared to sections from sporadic breast tumors with wild-type BRCA1. (v) Identification of high levels of prosaposin expression by the non-metastatic breast cancer cell line MDA-MB-231 as compared to the highly metastatic derivatives of this breast cancer cell line that metastasize to bone and lung. (vi) Identification of the secreted protein prosaposin as a regulator of thrombospondin-1 expression in stromal cells. Our studies have potential therapeutic implications to target specific genes that regulate the angiogenic switch, tumor mass expansion, and metastatic disease., The original document contains color images.

Phosphatidylserine (PS)-dependent erythrocyte adhesion to endothelium and sub-endothelial matrix components is mediated in part via thrombospondin (TSP). While TSP exhibits multiple cell-binding domains, the PS-binding site on TSP is unknown. Since a cell-binding domain for anionic heparin is located at the amino-terminus, we hypothesized that PS-positive red cells (PS+ve-RBCs) bind to this domain. We demonstrate that both heparin and its low-molecular-weight derivative enoxaparin (0.5-50u/ml) inhibited PS+ve-RBC adhesion to immobilized TSP in a concentration-dependent manner (21-77% inhibition, P<0.05). Pre-incubation of immobilized TSP with an antibody against the heparin-binding domain blocked PS+ve-RBC adhesion to TSP. Antibodies that recognize the collagen- and the carboxy-terminal CD47-binding domain on TSP had no effect on this process. While pre-incubation of PS+ve-RBCs with TSP-peptides from the heparin-binding domain containing the specific heparin-binding motif KKTRG inhibited PS+ve-erythrocyte adhesion to matrix TSP (P<0.001), these peptides in the immobilized form supported PS-mediated erythrocyte adhesion. A TSP-peptide lacking the binding-motif neither inhibited nor supported PS+ve-RBC adhesion. Additional experiments show that soluble-TSP also interacted with PS+ve-RBCs via its heparin-binding domain. Our results demonstrate that PS-positive erythrocytes bind to both immobilized and soluble TSP via its heparin-binding domain and that both heparin and enoxaparin, at clinically relevant concentrations, block this interaction. Other studies have shown that heparin inhibited P-selectin- and soluble-TSP-mediated sickle erythrocyte adhesion to endothelial cells. Our results taken together with the previously documented findings provide a rational basis for clinical use of heparin or its low-molecular-weight derivatives as therapeutic agents in treating vaso-occlusive pain in patients with sickle cell disease.

Our overall goal is to determine if human breast cancer can be prevented from becoming angiogenic when it is still at a microscopic size (< ~ 1 mm3). To date we have made the following progress: (1) We have cloned three different human breast cancers that undergo the angiogenic switch at predictable times. (2) We have found that the angiogenic switch time is modified by host stroma: two-fold earlier for tumors in the mammary fat pad, compared to tumors implanted in subcutaneous tissue. (3) We have found that the angiogenic switch is preceded by repression of stromal expression of thrombospodin-1. Angiogenic tumor cells continue to secrete a novel thrombospondin-1 repressing factor. This protein has been purified and partially sequenced. (4) For one of the breast cancers, the angiogenic switch can be detected at a microscopic size by a significant increase in bFGF in platelet alpha granules. (5) We have determined that the BRCA1 gene (breast cancer susceptibility gene), appears to regulate a ratio of thromobospondin-1 to VEGF in breast cancer cells. The lower the thrombospondin-1/VEGF ratio, the sooner the tumor cells will spontaneously switch to the angiogenic phenotype and grow large tumors in SCID mice., The original document contains color images.

Impaired wound healing and skin aging are characterized by neutral protease-mediated destruction of matrix macromolecules associated with disturbance in tissue repair. We synthesized a fatty acyl-peptide derivative at aims to simultaneously activate latent TGF-beta through its peptide domain, KFK, and inhibit MMPs through its lipophilic moiety, elaidic acid. Elaidyl-KFK as well as KFK were shown to activate LAP-TGF-beta both in vitro, using a solid phase assay with immobilized LAP-TGF-beta, and ex vivo using human dermal fibroblasts cultures. In both assays, as much as up to 10% of LAP-TGF-beta added could be recovered as active form. KQK, KQFK as well as their lipopeptide counterparts were inactive. Elaidyl-KFK-mediated LAP-TGF-beta activation led to up-regulation of collagen and TIMP-1 production and down regulation of PMA-induced MMP-1 expression in fibroblasts cultures. Those effects could be suppressed by supplementing cell culture medium with blocking TGF-beta antibody. Elaidyl-KFK inhibited MMP-2, MMP-9, MMP-3, MMP-1, in vitro with IC50 equal to 1.2, 1.0, 0.24 and 8.9 muM, respectively. Its ex vivo inhibitory capacity, as assessed using skin tissue sections, towards the elastin-degrading capacity of MMP-9 was even more pronounced. At a muM concentration, the lipopeptide decreased by up to 80% enzyme activity. Thus, "Lipospondin," i.e. elaidyl-KFK might be considered as a promising model compound to prevent age-associated dermal alterations. (C) 2004 Elsevier Inc. All rights reserved.

We have isolated a novel CSVTCG-specific tumor cell adhesion receptor and shown by immunohistochemical staining of human breast tumors that the receptor localizes to malignant ductal epithelium while no staining of epithelium in normal and benign tissues was observed. Preliminary studies suggest that the CSVTCG-specific receptor may function to promote the invasive behavior of breast epithelium and contribute to the development of malignancy. To test this hypothesis, we propose to transfect tall-length receptor cDNA in the sense or antisense orientation in order to either over-express or block receptor expression in breast carcinoma cell lines. We will use vector constructs that constitutively express the green fluorescent protein in order to enable localization of tumor cells in our animal studies.

Metastatic and primary tumor growth are both dependent on neovascularization. One can then predict that restrictions in the vascular supply to tumors could impinge on the expansion and uncontrolled growth of tumor cells. Studies presented in this proposal aim to evaluate the effect of an inhibitor of angiogenesis, thrombospondin-1 (TSP-1), in the growth of mammary tumors. The advantage of TSP-1 over other vascular inhibitors is that this protein is a natural angiogenic suppressor. TSP-1 is produced by the mammary gland during post-lactation and in fact contributes to the regression of capillaries during mammary gland involution. Therefore, we hypothesized that if effective in the regression of tumors, this molecule might not induce secondary or undesirable side effects. We have just completed two years of studies in this proposal. Our findings are encouraging; specifically, we were able to demonstrate in the lack of TSP-1 (utilizing TSP-1 null animals), neovascularization is significantly higher than in the presence of endogenous TSP-1. Furthermore, increase in the endogenous pool of TSP-1 induces apoptosis in capillary endothelial cells. The overall effect is a reduction in the vascular supply to the mammary gland and to the highly vascularized tumors. Experiments within the next two years of this proposal will aim to understand the molecular mechanism by which TSP-1 mediates endothelial cell apoptosis.