Background & aims: Advances in cross-sectional imaging have resulted in increased detection of intraductal papillary mucinous neoplasms (IPMNs) and their management remains controversial. At present, there is no reliable non-invasive method to distinguish between indolent and high risk IPMNs. We performed extracellular vesicle (EV) analysis to identify markers of malignancy in an attempt to better stratify these lesions.

Methods: Using a novel ultrasensitive digital extracellular vesicle screening technique (DEST) we measured putative biomarkers of malignancy (MUC1, MUC2, MUC4, MUC5AC, MUC6, Das-1, STMN1, TSP1, TSP2, EGFR, EpCAM, GPC1, WNT-2, EphA2, S100A4, PSCA, MUC13, ZEB1, PLEC1, HOOK1, PTPN6, and FBN1) in EV from patient-derived cell lines and then on circulating EV obtained from peripheral blood drawn from patients with IPMNs. We enrolled a total of 133 patients in two separate cohorts: a clinical discovery cohort (n=86) and a validation cohort (n=47).

Results: From 16 validated EV proteins in plasma samples collected from the discovery cohort, only MUC5AC showed significantly higher levels in high grade lesions. Of the 11 patients with invasive IPMN (inv/HG), 9 had high MUC5AC expression in plasma EV and of the 11 patients with high grade dysplasia alone, only 1 had high MUC5AC expression (specificity of 82%, sensitivity of 100%). These findings were corroborated in a separate validation cohort. The addition of MUC5AC as a biomarker to imaging and high risk stigmata allowed detection of all cases requiring surgery, whereas imaging and high risk stigmata alone would have missed 5/14 cases (36%).

Conclusions: MUC5AC in circulating EV can predict the presence of invasive carcinoma within IPMN. This approach has the potential to improve the management and follow-up of patients with IPMN including avoiding unnecessary surgery.

Keywords: : Pancreatic cancer; IPMN; early detection; exosome; pre-cancer.

Control of synapse number and function in the developing central nervous system is critical to the formation of neural circuits. Astrocytes play a key role in this process by releasing factors that promote the formation of excitatory synapses. Astrocyte-secreted thrombospondins (TSPs) induce the formation of structural synapses, which however remain post-synaptically silent, suggesting that completion of early synaptogenesis may require a two-step mechanism. Here, we show that the humoral innate immune molecule Pentraxin 3 (PTX3) is expressed in the developing rodent brain. PTX3 plays a key role in promoting functionally-active CNS synapses, by increasing the surface levels and synaptic clustering of AMPA glutamate receptors. This process involves tumor necrosis factor-induced protein 6 (TSG6), remodeling of the perineuronal network, and a β1-integrin/ERK pathway. Furthermore, PTX3 activity is regulated by TSP1, which directly interacts with the N-terminal region of PTX3. These data unveil a fundamental role of PTX3 in promoting the first wave of synaptogenesis, and show that interplay of TSP1 and PTX3 sets the proper balance between synaptic growth and synapse function in the developing brain.

BACKGROUND

Neointimal hyperplasia involves the migration of medial vascular smooth muscle cells (VSMCs) in response to arterial injury. Thrombospondin-1 (TSP1), platelet-derived growth factor (PDGF), and fibronectin (Fn) induce VSMC migration. Nitric oxide (NO) limits VSMC migration. The hypothesis of this study is that NO would dose dependently inhibit TSP1-induced, PDGF-induced, and Fn-induced VSMC chemotaxis.

METHODS

VSMCs were pretreated with serum free media or the NO donors diethylenetriamine NONOate or S-nitroso-N-acetyl-D,L-penicillamine. Chemotaxis to TSP1, PDGF, or Fn was determined. Analysis of variance with post hoc testing was done. P values < .05 were considered significant.

RESULTS

PDGF, TSP1, and Fn induced VSMC chemotaxis. NO donors inhibited chemotaxis of VSMCs to PDGF in a concentration-dependent manner. NO donors had a variable effect on TSP1-induced chemotaxis. NO donors did not inhibit Fn-induced chemotaxis.

CONCLUSIONS

The complex interactions of these proteins in vivo will need to be considered when developing NO-dependent therapies for neointimal hyperplasia.

Tumors depend upon angiogenesis for growth and metastasis. It is therefore critical to understand the inhibitory signaling mechanisms in endothelial cells that control angiogenesis. Epac is a cyclic adenosine 5'-monophosphate-activated guanine nucleotide exchange factor for Rap1. In this study, we show that activation of Epac or Rap1 leads to potent inhibition of angiogenesis in vivo. Epac/Rap1 activation down-regulates inhibitor of differentiation 1 (Id1), which negatively regulates thrombospondin-1 (TSP1), an inhibitor of angiogenesis. Consistent with this mechanism, activation of Epac/Rap 1 induces expression of TSP1; conversely, depletion of Epac reduces TSP1 levels in endothelial cells. Blockade of TSP1 binding to its receptor, CD36, rescues inhibition of chemotaxis or angiogenesis by activated Epac/Rap1. Mitogen-activated protein kinase kinase 5, a downstream mediator of vascular endothelial growth factor, antagonizes the effects of Epac/Rap1 by inducing Id1 and suppressing TSP1 expression. Finally, TSP1 is also secreted by fibroblasts in response to Epac/Rap1 activation. These results identify Epac and Rap1 as inhibitory regulators of the angiogenic process, implicate Id1 and TSP1 as downstream mediators of Epac/Rap1, and highlight a novel interplay between pro- and antiangiogenic signaling cascades involving multiple cell types within the angiogenic microenvironment.

Thrombospondins (TSPs) represent extracellular matrix (ECM) proteins belonging to the TSP family that comprises five members. All TSPs have a complex multidomain structure that permits the interaction with various partners including other ECM proteins, cytokines, receptors, growth factors, etc. Among TSPs, TSP1, TSP2, and TSP4 are the most studied and functionally tested. TSP1 possesses anti-angiogenic activity and is able to activate transforming growth factor (TGF)-β, a potent profibrotic and anti-inflammatory factor. Both TSP2 and TSP4 are implicated in the control of ECM composition in hypertrophic hearts. TSP1, TSP2, and TSP4 also influence cardiac remodeling by affecting collagen production, activity of matrix metalloproteinases and TGF-β signaling, myofibroblast differentiation, cardiomyocyte apoptosis, and stretch-mediated enhancement of myocardial contraction. The development and evaluation of TSP-deficient animal models provided an option to assess the contribution of TSPs to cardiovascular pathology such as (myocardial infarction) MI, cardiac hypertrophy, heart failure, atherosclerosis, and aortic valve stenosis. Targeting of TSPs has a significant therapeutic value for treatment of cardiovascular disease. The activation of cardiac TSP signaling in stress and pressure overload may be therefore beneficial.

BACKGROUND

TSP-1 is a vasoconstrictive protein, which is released from both endothelium and cardiomyocytes during ischemia and promotes platelet aggregation and adhesion to subendothelial layers in atherosclerotic lesions. During myocardial ischemia and reperfusion, TSP-1 disturbs local microcirculation by disrupting both NO-signaling as well as VEGF-pathways by activation of CD47 and CD36. Furthermore, activation of TGF-ß might induce excessive fibrosis after infarction. It was assumed that TSP-1 is washed out after successful coronary reperfusion. In this study, we examined circulating TSP-1 post emergency PCI as a risk factor for major adverse cardiac events after STEMI with and without ventricular fibrillation.

METHODS

TSP-1 levels in platelet poor plasma were measured in 54 patients after ST-elevation myocardial infarction. Major adverse cardiac events were monitored for 426 days.

RESULTS

Patients with decreased TSP levels after coronary stenting showed a significantly higher risk for MACE than patient with higher TSP levels (TSP-1[d0]: n = 46, no MACE = 16.38 ± 1.98 ug/mL vs. MACE 7.11 ± 1.54 ug/mL; p = 0.003). Kaplan-Meyer-analysis for MACE showed a better outcome above 10 ug/mL (p = 0.02). For MACE later than 3 months post-STEMI, the corresponding Kaplan-Meier-analysis yielded a p-value of 0.01. The number needed to diagnose for late MACE was 2.158.

CONCLUSIONS

Low plasma levels of TSP1 after PCI are associated with MACE. Due to its procoagulant effects and dysregulation of microvascular tone, adequately powered prospective studies are warranted to test the impact of TSP-1 on cardiac microcirculation, endothelial function and remodeling. TSP-1 might serve as a new diagnostic and therapeutic approach in cardiovascular disease.

ADAM12, (ADisintegrin and metalloproteinase domain-containing protein 12), is upregulated in epithelial cancers and contributes to increased tumor proliferation, metastasis, and endocrine resistance. However, its role in tumor angiogenesis is unknown. Here, we report that ADAM12 is upregulated in the vessels of aggressive breast tumors and exerts key regulatory functions. ADAM12 significantly increases bFGF-mediated angiogenesis in vivo and ADAM12 levels are upregulated in tumors that have undergone a switch to the angiogenic phenotype. Importantly, ADAM12-overexpressing breast tumors display a higher microvessel density (MVD). Our goal was to identify the mechanisms by which tumor-associated ADAM12 promotes angiogenesis. ADAM12 expression in breast tumor cells correlated with a significant upregulation of proangiogenic factors such as VEGF and MMP-9 and downregulation of antiangiogenic factors such as Thrombospondin-1 (THBS1/TSP1) and Tissue Inhibitor of Metalloproteinases-2 (TIMP-2). Co-culture with ADAM12-expressing tumor cells promoted endothelial cell (EC) recruitment and capillary tube formation. Conversely, downregulation of endogenous ADAM12 in breast cancer cell lines resulted in reduction of pro-angiogenic factors and EC recruitment. These ADAM12-mediated effects are driven by the activation of EGFR, STAT3 and Akt signaling. Blockade of EGFR/STAT3 or silencing of ADAM12 reversed the proangiogenic tumor phenotype, significantly downregulated pro-angiogenic mitogens and reduced EC recruitment. In human breast cancer tissues, ADAM12 expression was significantly positively correlated with pro-angiogenic factors including VEGF and MMP-9 but negatively associated with TSP1.Implications: These novel findings suggest that ADAM12 regulates EC function and facilitates a proangiogenic microenvironment in a STAT3-dependent manner. A combined approach of targeting ADAM12 and STAT3 signaling in breast cancer may represent a promising strategy to inhibit tumor neovascularization. Mol Cancer Res; 15(11); 1608-22. ©2017 AACR.

Objective: Abdominal aortic aneurysm is characterized by the progressive loss of aortic integrity and accumulation of inflammatory cells primarily macrophages. We previously reported that global deletion of matricellular protein TSP1 (thrombospondin-1) protects mice from aneurysm formation. The objective of the current study is to investigate the cellular and molecular mechanisms underlying TSP1's action in aneurysm. Approach and Results: Using RNA fluorescent in situ hybridization, we identified macrophages being the major source of TSP1 in human and mouse aneurysmal tissues, accounting for over 70% of cells that actively expressed Thbs1 mRNA. Lack of TSP1 in macrophages decreased solution-based gelatinase activities by elevating TIMP1 (tissue inhibitor of metalloproteinases-1) without affecting the major MMPs (matrix metalloproteinases). Knocking down Timp1 restored the ability of Thbs1^-/- macrophages to invade matrix. Finally, we generated Thbs1 ^flox/flox mice and crossed them with Lyz2-cre mice. In the CaCl₂-induced model of abdominal aortic aneurysm, lacking TSP1 in myeloid cells was sufficient to protect mice from aneurysm by reducing macrophage accumulation and preserving aortic integrity.

Conclusions: TSP1 contributes to aneurysm pathogenesis, at least in part, by suppressing TIMP1 expression, which subsequently enables inflammatory macrophages to infiltrate vascular tissues.

Keywords: aortic aneurysm, abdominal; gelatinase; human; inflammation; macrophages.

Prostate cancer metastasis to bone is terminal; thus, novel therapies are required to prevent end-stage disease. Kallikrein-related peptidase 4 (KLK4) is a serine protease that is overproduced in localized prostate cancer and is abundant in prostate cancer bone metastases. In vitro, KLK4 induces tumor-promoting phenotypes; however, the underlying proteolytic mechanism is undefined. The protein topography and migration analysis platform (PROTOMAP) was used for high-depth identification of KLK4 substrates secreted by prostate cancer bone metastasis-derived PC-3 cells to delineate the mechanism of KLK4 action in advanced prostate cancer. Thirty-six putative novel substrates were determined from the PROTOMAP analysis. In addition, KLK4 cleaved the established substrate, urokinase-type plasminogen activator, thus validating the approach. KLK4 activated matrix metalloproteinase-1 (MMP1), a protease that promotes prostate tumor growth and metastasis. MMP1 was produced in the tumor compartment of prostate cancer bone metastases, highlighting its accessibility to KLK4 at this site. KLK4 further liberated an N-terminal product, with purported angiogenic activity, from thrombospondin-1 (TSP1) and cleaved TSP1 in an osteoblast-derived matrix. This is the most comprehensive analysis of the proteolytic action of KLK4 in an advanced prostate cancer model to date, highlighting KLK4 as a potential multifunctional regulator of prostate cancer progression.

Reciprocal interactions between cancers and the surrounding microenvironment have an important role in tumour evolution. In this study, our data suggested that through thrombospondin 1 (TSP1), tumour-associated microvessel provides a dormant niche to sustain inactive status of breast invasive ductal carcinoma (IDC) cells. TSP1 levels in the tumour stroma were negatively correlated with vascular indoleamine 2,3-dioxygenase 1 (IDO1) in IDC tissues. IDO1 is an intracellular enzyme initiating the first and rate-limited step of tryptophan breakdown. Lower stromal TSP1 levels and positive tumour vascular IDO1 staining seems to associate with poor survive of patients with IDC. IDC cells induced a significantly increase in IDO1 expression in endothelial cells (ECs). IFNγ exerts a similar effect on ECs. We hypothesized a tryptophan starvation theory that since tryptophan is essential for the synthesis of TSP1, IDO1 induce a decrease in tryptophan availability and a reduction in TSP1 synthesis in ECs, leading to overcoming the dormancy state of IDC cells and exacerbating conditions such as tumour invasion and metastasis. These findings identify a non-canonical role of IFNγ/IDO1/TSP1 axis in microvascular niche-dominated dormancy of breast invasive ductal carcinoma with a solid foundation for further investigation of therapeutic and prognostic relevance.

Immunoglobulin Gs (IgGs) against ADAMTS13 are major causes of acquired (idiopathic) thrombotic thrombocytopenic purpura (TTP). We report here a novel cell-based assay using glycosylphosphatidylinositol (GPI)-anchored ADAMTS13 or variants expressed on cell membrane for assessment of autoantibodies in patients with TTP. We showed that IgGs from all 26 patients with acquired TTP bound to cells expressing a GPI anchored full-length ADAMTS13 (gFL) and a variant truncated after the spacer domain (gS). Also, IgGs from 25/26 (96.7%) of these TTP patients bound to cells expressing a GPI-anchored C-terminal fragment, TSP1 2-8 plus CUB (gT2C). In contrast, none of the 20 healthy blood donors showed detectable binding of their IgGs to the cells expressing gFL, gS, and gT2C. A moderate, but statistically significant correlation was observed between plasma concentrations of anti-ADAMTS13 IgG and positive cells expressing gFL (r=0.65), gS (r=0.67), and gT2C (r=0.42). These results suggest that the microtiter-plate assay and the cell-based assay may detect differential antigenic epitopes. Moreover, antigens clustered on cell membranes may enhance antibody binding affinity, thereby increasing analytical sensitivity. Finally, our assay was able to determine kinetic changes of plasma levels of anti-ADAMTS13 IgGs in TTP patients during plasma therapy. Together, our findings suggest that the novel cell-based assay may be applicable for rapid identification and mapping of anti-ADAMTS13 autoantibodies in patients with acquired TTP.

The development of a hyperexcitable neuronal network is thought to be a critical event in epilepsy. Thrombospondins (TSPs) regulate synaptogenesis by binding the neuronal α2δ subunit of the voltage-gated calcium channel. TSPs regulate synapse formation during development and in the mature brain following injury. It is unclear if TSPs are involved in hyperexcitability that contributes to the development of epilepsy. Here we explore the development of epilepsy using a pentylenetetrazole (PTZ) kindling model in mice lacking TSP1 and TSP2. Unexpectedly, we found increased sensitivity to PTZ kindling in mice lacking TSP1, while mice lacking TSP2 kindled similar to wild-type. We found that the increased seizure susceptibility in the TSP1 knockout (KO) mice was not due to a compensatory increase in TSP2 mRNA as TSP1/2 KO mice were sensitive to PTZ, similar to the TSP1 KO mice. Furthermore, there were similar levels of TGF-B signal activation during kindling in the TSP1 KO mice compared to wild-type. We observed decreased expression of voltage-dependent calcium channel subunit CACNA2D1 mRNA in TSP1, TSP2, and TSP1/2 KO mice. Decreased CACNA2D2 mRNA was only detected in mice that lacked TSP1 and α2δ-1/2 protein levels in the cortex were lower in the TSP 1/2 KO mice. CACNA2D2 knockout mice have spontaneous seizures and increased PTZ seizure susceptibility. Here we report similar findings, TSP1, and TSP1/2 KO mice have low levels of CACNA2D2 mRNA expression and α2δ-1/2 receptor level in the cortex, and are more susceptible to seizures. CACNA2D2 mutations in mice and humans can cause epilepsy. Our data suggest TSP1 in particular may control CACNA2D2 levels and could be a modifier of seizure susceptibility.

Sulfatide (galactocylceramide-3'-sulfate), a cell surface glycosphingolipid interacts with several cell adhesion molecules including fibrinogen, von Willebrand factor (VWF), P-selectin, thrombospondin (TSP) and laminin, which are involved in haemostasis. We have used a sulfatide-specific single-chain fragment variable (scFv) antibody probe PA38 and sulfatide-deficient mice to investigate the role of membrane sulfatide in platelet function. PA38 bound to platelets and binding increased following platelet activation. Sulfatide was localized as a large cluster towards the center of the platelet surface when examined in a confocal microscope. PA38 (20 microg/ml) inhibited the adhesion of activated platelets to fibrinogen, VWF, P-selectin, TSP1 and laminin by 30%, 30%, 75%, 20% and 35%, respectively, compared to a control scFv (p < 0.05). Furthermore, PA38 inhibited collagen, ADP, thrombin and ristocetin-induced platelet aggregation in PRP by 25%, 30%, 18% and 20%, respectively, compared to the control scFv (p < 0.05). In a PFA-100 platelet function assay, PA38 prolonged the occlusion time by 25% (p < 0.05). Under flow PA38 decreased the thrombus formation on collagen by 31%, (p < 0.01). Sulfatide-deficient mice displayed an extended lag-phase in collagen-induced platelet aggregation compared to wild type (p < 0.05), though in-vivo haemostasis did not differ significantly. Thus, this study provides new evidence for a role for membrane sulfatide in platelet function.

The prothrombotic fibrin clot phenotype has been reported in patients with thrombotic antiphospholipid syndrome (APS) and venous thromboembolism (VTE). Protein composition of plasma fibrin clots in APS has not been studied. We evaluated 23 patients with thrombotic APS, 19 with VTE alone, and 20 well-matched controls. A proteomic analysis of fibrin clots generated from citrated plasma was based on liquid chromatography-mass spectrometry. Plasma levels of thrombospondin-1 (TSP1), apolipoprotein(a), A-I, and B-100, complement components (C)3a, C5b-C9, histidine-rich glycoprotein (HRG), and prothrombin were evaluated using immunoenzymatic tests. In plasma fibrin clots of APS patients, compared with VTE subjects and controls, we identified decreased amounts of (pro)thrombin, antithrombin-III, apolipoprotein A-I, and HRG with no differences in plasma levels of antithrombin, prothrombin, along with lower plasma HRG and apolipoprotein A-I. In APS patients, plasma HRG positively correlated with amounts of clot-bound HRG, while apolipoprotein A-I was inversely associated with clot-bound levels of this protein. The most predominant proteins within the clots of APS patients were bone marrow proteoglycan, C5-C9, immunoglobulins, apolipoprotein B-100, platelet-derived proteins, and TSP1. Our study is the first to demonstrate differences in the protein composition of fibrin clots generated from plasma of thrombotic APS patients versus those with VTE alone.

Mutation of c-K-ras oncogene is an important step in progression of colon cancer. We used a hammerhead ribozyme (KrasRz) against mutated K-ras gene transcripts (codon 12, GTT) to inactivate mutant K-ras function in the colon cancer cell line SW480, harbouring a mutant K-ras gene. The beta-actin promoter-driven KrasRz sequence (pHbeta/KrasRz) was introduced into these cells (SW480/KrasRz), and we evaluated its effects on growth of the colon cancer. The gene expression of angiogenesis-related molecules (vascular endothelial growth factor and thrombospondin) was also estimated in SW480/KrasRz. KrasRz specifically and efficiently cleaved the mutant K-ras mRNA but not wild-type mRNA in vitro. SW480/KrasRz showed decreased growth rate under tissue culture conditions (P< 0.01, Dunnett's test). The xenotransplantability of SW480/KrasRz (XeSW480/KrasRz) was significantly decreased in nude mice (P< 0.05, Fisher's exact test). Tumour volume of the xenografts XeSW480/KrasRz was significantly smaller than that of XeSW480/DisKrasRz (P< 0.01, Dunnett's test). Gene expression of VEGF was suppressed in SW480/KrasRz, while TSP1 gene expression was enhanced. The SW480/KrasRz cells showed apoptosis-related features including nuclear condensation and DNA fragmentation. These results suggested that the hammerhead ribozyme-mediated inactivation of the mutated K-ras mRNA induced growth suppression, apoptosis and alteration of angiogenic factor expression.

The purpose of this study was to investigate the changes that occur in the lacrimal glands (LGs) in female thrombospondin 1 knockout (TSP1-/-) mice, a mouse model of the autoimmune disease Sjogren's syndrome. The LGs of 4, 12, and 24 week-old female TSP1-/- and C57BL/6J (wild type, WT) mice were used. qPCR was performed to measure cytokine expression. To study the architecture, LG sections were stained with hematoxylin and eosin. Cell proliferation was measured using bromo-deoxyuridine and immunohistochemistry. Amount of CD47 and stem cell markers was analyzed by western blot analysis and location by immunofluorescence microscopy. Expression of stem cell transcription factors was performed using Mouse Stem Cell Transcription Factors RT2 Profiler PCR Array. Cytokine levels significantly increased in LGs of 24 week-old TSP1-/- mice while morphological changes were detected at 12 weeks. Proliferation was decreased in 12 week-old TSP1-/- mice. Three transcription factors were overexpressed and eleven underexpressed in TSP1-/- compared to WT LGs. The amount of CD47, Musashi1, and Sox2 was decreased while the amount of ABCG2 was increased in 12 week-old TSP1-/- mice. We conclude that TSP1 is necessary for maintaining normal LG homeostasis. Absence of TSP1 alters cytokine levels and stem cell transcription factors, LG cellular architecture, decreases cell proliferation, and alters amount of stem cell markers.

Liquid chromatography using a tandem mass spectrometer (LC-MS/MS) is a method of proteomic analysis. A shotgun analysis by LC-MS/MS comprehensively identifies proteins from tissues and cells with high resolution. The hepatic function of mice with acute hepatitis following the intraperitoneal administration of CCL4 was improved by the tail vein administration of the culture conditional medium (CM) of human mesenchymal stem cells from adipose tissue (hMSC-AT). In this study, a secreted protein expression analysis of hMSC-AT was performed using LC-MS/MS; 128 proteins were identified. LC-MS/MS showed that 106 new functional proteins and 22 proteins (FINC, PAI1, POSTN, PGS2, TIMP1, AMPN, CFAH, VIME, PEDF, SPRC, LEG1, ITGBL, ENOA, CSPG2, CLUS, IBP4, IBP7, PGS1, IBP2, STC2, CTHR1, CD9) were previously reported in hMSC-AT-CMs. In addition, various proteins associated with growth (SAP, SEM7A, PTK7); immune system processes (CO1A2, CO1A1, CATB, TSP1, GAS6, PTX3, C1 S, SEM7A, G3P, PXDN, SRCRL, CD248, SPON2, ENPP2, CD109, CFAB, CATL1, MFAP5, MIF, CXCL5, ADAM9, CATK); and reproduction (MMP2, CATB, FBLN1, SAP, MFGM, GDN, CYTC) were identified in hMSC-AT-CMs. These results indicate that a comprehensive expression analysis of proteins by LC-MS/MS is useful for investigating new factors associated with cellular components, biological processes, and molecular functions.

The CCN family of proteins typically consists of four distinct peptide domains: an insulin-like growth factor binding protein-type (IGFBP) domain, a Von Willebrand Factor C (VWC) domain, a thrombospondin type 1 repeat (TSP1) domain, and a carboxy-terminal (CT) domain. The six family members participate in many processes, including proliferation, motility, cell-matrix signaling, angiogenesis, and wound healing. Accumulating evidence suggests that truncated and alternatively spliced isoforms are responsible for the diverse functions of CCN proteins in both normal and pathophysiologic states. Analysis of the properties and functions of individual CCN domains further corroborates this idea. CCN5 is unique among the CCN family members because it lacks the CT-domain. To dissect the domain functions of CCN5, we are developing domain-specific mouse monoclonal antibodies. Monoclonal antibodies have the advantages of great specificity, reproducibility, and ease of long-term storage and production. In this communication, we injected mixtures of GST-fused rat CCN5 domains into mice to generate monoclonal antibodies. To identify the domains recognized by the antibodies, we constructed serial expression plasmids that express dual-tagged rat CCN5 domains. All of the monoclonal antibodies generated to date recognize the VWC domain, indicating it is the most highly immunogenic of the CCN5 domains. We characterized one particular clone, 22H10, and found that it recognizes mouse and rat CCN5, but not human recombinant CCN5. Purified 22H10 was successfully applied in Western Blot analysis, immunofluorescence of cultured cells and tissues, and immunoprecipitation, indicating that it will be a useful tool for domain analysis and studies of mouse-human tumor models.

Angiogenesis, the formation of new blood capillaries from pre-existing vessels, is a hallmark of cancer. Thus far, strategies for reducing tumor angiogenesis have focused on inhibiting pro-angiogenic factors, while less is known about the therapeutic effects of mimicking the actions of angiogenesis inhibitors. Thrombospondin-1 (TSP1) is an important endogenous inhibitor of angiogenesis that has been investigated as an anti-angiogenic agent. TSP1 impedes the growth of new blood vessels in many ways, including crosstalk with pro-angiogenic factors. Due to the complexity of TSP1 signaling, a predictive systems biology model would provide quantitative understanding of the angiogenic balance in tumor tissue. Therefore, we have developed a molecular-detailed, mechanistic model of TSP1 and vascular endothelial growth factor (VEGF), a promoter of angiogenesis, in breast tumor tissue. The model predicts the distribution of the angiogenic factors in tumor tissue, revealing that TSP1 is primarily in an inactive, cleaved form due to the action of proteases, rather than bound to its cellular receptors or to VEGF. The model also predicts the effects of enhancing TSP1's interactions with its receptors and with VEGF. To provide additional predictions that can guide the development of new anti-angiogenic drugs, we simulate administration of exogenous TSP1 mimetics that bind specific targets. The model predicts that the CD47-binding TSP1 mimetic dramatically decreases the ratio of receptor-bound VEGF to receptor-bound TSP1, in favor of anti-angiogenesis. Thus, we have established a model that provides a quantitative framework to study the response to TSP1 mimetics.

In the search for androgen target genes responsible for malignant growth in S115 mouse mammary tumor cells we found that thrombospondin 1 (TSP1) expression was strongly downregulated by testosterone (Te). Experiments with cycloheximide suggested that Te repression of TSP1 was dependent on de novo protein synthesis. TSP1 repression by Te was preceded by the induction of fibroblast growth factor 8 (FGF8) expression. FGF8 has previously been shown to mediate androgen effects on proliferation of S115 cells by autocrine/paracrine mechanisms. It has also been shown to increase breast cancer cell growth as tumors in nude mice and to stimulate tumor angiogenesis. We studied here the possibility that FGF8 belonged to the Te-induced de novo synthesized proteins that mediate the effect of Te on TSP1 expression in these cells. We found that addition of FGF8b to in vitro cultures or ectopic expression of FGF8b in S115 cells repressed TSP1 expression at mRNA and protein levels even in the absence of Te. FGF2, another angiogenic member of FGF family, also downregulated TSP1 mRNA level in the in vitro cultures of S115 cells. The antisense oligonucleotides for FGF8 did not, however, prevent Te-repression of TSP1 mRNA expression and a neutralizing anti-FGF8b antibody only partially opposed Te induced downregulation of TSP1. These results suggest that both androgen and FGF8 inhibit TSP1 expression independently. They also suggest that opposite to many other androgen-induced responses in S115 cells, the effect of Te on the expression TSP1 is not mediated by FGF8.

Three fluorescent probes were constructed by incorporating an α,β-unsaturated ketone to a coumarin fluorophore. The selective addition of sulfite to the alkene of TSP assisted by cetyltrimethyl ammonium bromide (CTAB) micelle can be visualized by dramatic color and ratiometric fluorescence changes. In CTAB-PBS system, the fluorescence intensity ratio at 465 nm and 592 nm (I465/I592) and the absorbance ratio at 390 nm and 470 nm (A390/A470) were linearly proportional to sulfite concentration in the range of 0.5-150 μM, and the detection limit was 0.2 μM. Good selectivity and competition of TSP1 towards sulfite over several anions and biological thiols were acquired. Probe TSP1 was used to detect sulfite in three realistic samples (mineral water, sugar and white wine) with good recovery.

Transmission of human cytomegalovirus (HCMV) to the fetus is the most common type of intrauterine infection; the mechanism of HCMV pathogenesis in the developing central nervous system remains unclear. Thrombospondins 1 and 2 (TSP1, TSP2) produced by immature astrocytes are critical for fetal synaptogenesis. To examine the effect of HCMV on fetal astrocytes, human fetal astrocytes were isolated and cultured with HCMV AD169. Cells were harvested at different time points. Protein and mRNA expressions of TSP1 and TSP2 were determined using RT-qPCR, western blotting analysis, and enzyme-linked immunosorbent assay. The results showed that HCMV infection induced time-dependent decreases in mRNA and protein expressions of both TSP1 and TSP2 in astrocytes. Flow cytometry was used to detect apoptosis of HCMV-infected astrocytes, and the result indicated that there was no linkage between cell apoptosis and the decrease in TSP1 and TSP2 expressions induced by HCMV infection. When ganciclovir treatment was performed on HCMV-infected astrocytes, results showed that ganciclovir treatment inhibited the reduction of TSP1 and TSP2 expression in astrocytes. In the further study, pEGFP-N3-IE1 was transfected into astrocytes to identify that it was not IE1 but active viral replication that was essential in the continuous decrease of TSP1 and TSP2 expressions in HCMV-infected astrocytes.

Our objective was to examine the topology-, gestation- and labor-related changes of estrogen receptor (ER)alpha, progesterone receptor (PR), oxytocin receptor (OTR) and thrombospondin-1 (TSP1) mRNA in pregnant baboon myometrium. ER alpha, PR, OTR and TSP1 mRNAs extracted from the lower uterine segment and fundal myometrium of pregnant baboons not in labor between 121 and 180 days of gestational age (n=9) and in established spontaneous labor between 164 and 193 days of gestational age (n=5) were analyzed by Northern blot. There were no topology-, gestation- or labor-related changes of ER alpha and PR mRNA in or between the lower uterine segment or/and the fundus. OTR mRNA was the same in the lower uterine segment and the fundus from baboons not in labor and non-labor fundal, but not lower uterine segment, myometrial OTR mRNA increased with gestation (R(2)=0.81, P<0.05). Fundal OTR mRNA rose significantly compared with the lower uterine segment during spontaneous labor. TSP1 mRNA increased significantly in both the fundus and lower uterine segment during labor. TSP1 mRNA in the lower uterine segment during spontaneous labor was higher than in the fundus during spontaneous labor. In conclusion, fundal and lower uterine segment ER alpha and PR mRNA remained unchanged in late gestation and spontaneous labor. The increased OTR mRNA may serve as a mechanism to increase uterine sensitivity to OT during late gestation. The higher fundal OTR mRNA compared with the lower uterine segment provides polarity which assists fetal expulsion by uterine contractions during labor. The significance of increased TSP1 mRNA during labor may relate to homeostasis and merits further study.

Thrombospondin-1 (TSP1) plays a role in the immune tolerance, and is involved in the pathogenesis of glioma. This study aims to investigate the role of the glioma-derived TSP1 in the induction of the tumor immune tolerance. The results showed that the primary human glioma cells expressed high levels of TSP1. Glioma cells enhanced the expression of transforming growth factor (TGF)-β in CD4⁺ CD16⁻ naïve monocytes (Mos). The TGF-β⁺ Mos showed inhibitory effect on CD8⁺ T cell proliferation. We conclude that glioma cell-derived TSP1 facilitates the induction of TGF-β in Mos. The TSP1 may be a potential therapeutic target of glioma.