TSP-1 Expression Research Articles

Abstract A195: Inhibition of angiogenesis and growth of HER2-overexpressing tumors by SV40 T/t-common polypeptide

Abstract HER2 overexpression in various tumors results in increased angiogenesis, metastasis and reduced survival. Previously we reported that simian virus 40 T/t-common polypeptide can repress HER2 expression and specifically induce apoptosis in HER2-overexpressing cancer cells. In this study, we further explored the effect of T/t-common on tumor angiogenesis. HER2-overexpressing cancer cells were either infected with T/t-common-carrying adenovirus (rAd-T/t) or vector virus (rAd-V). The supernatant of infected cells were then used in the Tanswell migration, tube formation, and Matrigel plug assays to evaluate its ability to induce angiogenesis. Our data indicate that conditioned medium from rAd-T/t-infected cells had much lower ability to induce the migration and tube formation of endothelial cells and to induce blood vessel formation in Matrigel plugs than that from rAd-V-infected cells. T/t-common was found to be able to inhibit the expression of pro-angiogenic factor VEGF-A through inhibiting the HER2-ERK-HIF-1 signaling pathway. Moreover, T/t-common could induce the expression of anti-angiogenic factor TSP-1 through activating the HER2-p38 pathway. Finally we show that infection of HER2-overexpressing SK-OV-3 xenograft tumors by rAd-T/t virus could lead to inhibition of tumor growth and microvessel formation in NOD/SCID mice. Together, these data suggest that T/t-common is a potential gene therapeutic agent that can inhibit the growth and angiogenesis of HER2-overexpressing tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A195.

Expression of TSP-1 in the endometrial adenocarcinoma and its correlation with VEGF and CD105

Objective To study the expression of inhibitors of DNA binding-1(Id-1)in endometrial adenocarcinoma and its correlation with VEGF,TSP-1.Methods By using immunohistochemistry and image analysis,the expressions of Id-1,VEGF and TSP-1 proteins were studied in a group of 56 patients with endometrial adenocarcinoma,18 patients with atypia hyperplasia and 20 patients with normal endometria.Results The expression of Id-1 in endometrial adenocarcinoma tissues was significantly higher than that in normal endometria and atypia hyperplasia tissues(P0.05)and was significantly correlated with the histological grades,depth of myometrial invasion and clinica1 stage(all P0.05).Id-1 expression in endometrial adenocarcinoma tissues was positively correlated with VEGF expression(r=0.318,P=0.017)and negatively correlated with TSP-1 expression(r=-0.453,P=0.000).No significant association was found between Id-1 expression and patients' survival time.Conclusions Overexpression of Id-1 is common in endometrial adenocarcinoma.Id-1 might be related to the carcinogenesis and progression of endometrial adenocarcinoma by decreasing TSP-1 expression and increasing VEGF expression.

Inhibition of angiogenesis of LCI-D20 hepatocellular carcinoma by metronomic chemotherapy of S-1

To investigate the role of metronomic chemotherapy of S-1 on angiogenesis of hepatocellular carcinoma in animal model. S-1 was dissolved in a 0.5% (w/v) HPMC solution. 30 LCI-D20 were randomly devided into five groups: control group(O), 10 mg * kg(-1) * d(-1) S-1 group (A), 1 mg * kg(-1) * d(-1) S-1 group (B), 0.5 mg * kg(-1) * d(-1) S-1 group (C) and 0.25 mg * kg(-1) * d S-1 group (D). 28 days after the treatment with 0.5% (w/v) HPMC solution, tumors in LCI-D20 mice were moved out. Tumor mass was measured and microvessel density (MVD) was used to evaluate angiogenesis in tumor. The cellular apoptosis was determined using flow cytometry. The expression of VEGF, bFGF and TSP-1 was measured by RT-PCR. The mean tumor mass was 2.01, 0.38, 1.12, 1.38, 2.27 g in O, A, B, C, D group, respectively. The mean MVD was 39.57, 19.90, 5.93, 17.10, 29.53 in O, A, B, C, D respectively. The mean tumor cellular apoptosis rate was 4.08%, 44.37%, 31.73%, 19.83%, and 8.25% in O, A, B, C, D respectively. The expression of VEGF and bFGF in O group was highest, and A was slightly low, and C and D taked the third place, and B was the lowst; The expression of TSP-1 in B was highest, and C and D were slightly low, and A taked the third place, and O was the lowst. Metronomic chemotherapy of S-1 destabilizes pre-existing tumor vasculature and inhibits ongoing angiogenesis.

Cancerous, but not stromal, thrombospondin-2 contributes prognosis in pulmonary adenocarcinoma

Thrombospondin (TSP)-2 is known to be an endogenous negative regulator of vascularization in human cancer. However, it is unclear whether TSP-2 expression is related to neovascularization and prognosis in non-small cell lung cancer. In this study, we quantitatively examined the expression of TSP-2 mRNA by real-time reverse transcription-polymerase chain reaction (RT-PCR) in 102 pulmonary adenocarcinomas. All 102 carcinoma specimens expressed TSP-2 mRNA. The expression of TSP-2 mRNA in carcinoma was significantly higher than normal lung tissues (p<0.0001, Kruskal-Wallis test). Sizes of tumors were significantly correlated with TSP-2 gene expression (p=0.0179, Kruskal-Wallis test). The TSP-2 expression levels of the stage II/III pulmonary carcinomas were significantly increased as compared to those of stage I (p=0.0136, Kruskal-Wallis test). Thirty-five patients with high TSP-2 mRNA expression showed poor prognosis in survival (p=0.0139, log-rank test). We examined TSP-2 protein localizations in the pulmonary adenocarcinoma overexpressing TSP-2 mRNA. The TSP-2 localizations were categorized in two patterns: cancerous TSP-2 expression pattern (TSP-2 expression in the cancerous cells) and non-cancerous TSP-2 expression pattern (TSP-2 expression in the stromal lymphoid cells). Pulmonary adenocarcinoma patients with cancerous TSP-2 expression pattern showed good prognosis (p=0.0322; Fisher's probability exact test). Pulmonary adenocarcinoma patients with non-cancerous TSP-2 expression pattern showed poor prognosis (p=0.0220; Fisher's probability exact test). Non-cancerous TSP-2 expressions may reflect secondary reactions in the cancerous stroma. The stromal TSP-2 expression is not enough to suppress growth of pulmonary adenocarcinoma, while the cancerous TSP-2 expression directly inhibits growth of the carcinoma.

Differential Expression of Pancreatitis-Associated Protein and Thrombospondins in Arterial versus Venous Tissues

Background/Aims: Arteries and veins modulate cardiovascular homeostasis and contribute to hypertension pathogenesis. Functional differences between arteries and veins are based upon differences in gene expression. To better characterize these expression patterns, and to identify candidate genes that could be manipulated selectively in the venous system, we performed whole genome expression profiling of arteries and veins. Methods: We used the CodeLink platform and the major artery (thoracic aorta) and vein (caudal vena cava) of the rat. Results: The most prominent difference was pancreatitis-associated protein (PAP1), expressed 64-fold higher in vena cava versus aorta. Expression of mRNA for thrombospondins (TSP-1, TSP-4) was greater than 5-fold higher in veins versus arteries. Higher mRNA expression of TSP-1, TSP-2, TSP-4 and PAP1 in vena cava versus aorta was confirmed by PCR. Immunohistochemical analysis of tissue sections qualitatively confirmed a higher expression of these proteins in vena cava versus aorta. Conclusion: This is the first gene array study of adult rat arterial and venous tissues, and also the first study to report differences in inflammatory genes between arteries and veins. Data from these studies may provide novel insights into the genetic basis for functional differences between arteries and veins in health and disease.

Thrombospondin-1–Mediated Regulation of Microglia Activation after Retinal Injury

Thrombospondin (TSP)-1 has been demonstrated to play a vital role in immune privilege. The functional phenotype of ocular antigen-presenting cells that contributes to the immune privilege status of the eye is dependent on their expression of TSP-1. Microglia, the local antigen-presenting cells in the retina, undergo rapid activation in response to injury and have the ability to produce both proinflammatory and regenerative neurotrophic factors. In this study, the authors examined TSP-1 as a potential regulator of these phenotype of microglia activated in response to retinal injury. Expression of markers associated with activated microglia were examined by immunofluorescent staining and semiquantitative real-time PCR analysis of retina derived from WT or TSP-1 null mice at various time intervals after light- or laser-induced retinal injury. In the absence of TSP-1, microglia in uninjured retina express major histocompatibility complex class II and migrate to the outer layers of the retina. Constitutively increased expression of activated microglia-derived inflammatory molecules such as TNF-alpha and iNOS is detectable in TSP-1 null retina compared with WT controls. After both light-induced and laser-induced retinal injury, enhanced migration of microglia is detected in TSP-1 null retina, and these microglia express markers associated with a proinflammatory phenotype. Compared with WT retina, TSP-1 null retina fails to recover from the laser-induced injury, resulting in irreversible damage. TSP-1 supports an anti-inflammatory phenotype of microglia in the retina and promotes recovery from injury.

A phase I study of ABT 510 and concurrent temozolamide and radiotherapy for patients with newly diagnosed glioblastoma multiforme

2023 Background: ABT-510 (Abbott Laboratories, Abbott Park, IL, USA) is a Thrombospondin-1 (TSP-1) mimetic drug with anti-angiogenic properties. This phase I dose escalation trial was designed to study the maximum tolerated dose (MTD) of ABT 510 when used concurrently with temozolomide (TMZ) and radiotherapy (RT) in patients with newly diagnosed glioblastoma multiforme (GBM). Methods: A total of 23 patients with newly diagnosed, histologically verified GBM were enrolled between April 2005 and January 2007, after obtaining written consent. The study was approved by the University of Alabama at Birmingham (UAB) Institutional Review Board. Four cohorts with three patients in each, receiving subcutaneous ABT 510 injection at doses of 20, 50, 100, and 200 mg/day were studied. The starting dose was primarily based on preclinical findings from animal studies and phase I studies on healthy subjects and cancer patients. Treatment plan included 10 weeks of induction phase (TMZ and RT with ABT 510) followed by a maintenance phase (ABT 510 and TMZ) of 14 cycles each consisting of 28 days. Patients were monitored with brain MRI along with laboratory values for dose limiting toxicities (DLT) defined as grades 3–4 non-hematological toxicities and grade 4 hematological toxicities (neutropenia or thrombocytopenia). In the absence of a DLT in at least two of the three patients, the dose was increased by 50% in the next cohort of patients. Therapy was discontinued if 14 maintenance cycles were completed, disease progression occurred, or if the patient requested withdrawal. Disease progression and survival statistics were analyzed. Results: During this trial, grade 3/4 DLT were not observed even after the dose was increased to 200 mg/day, hence, the last cohort was expanded to include 14 patients. A MTD was not defined. The median time to tumor progression (TTP) was 220 days and the median overall survival was 422 days. Gene expression analysis of the tumor pathology will be performed to evaluate the relationship between the expression of TSP-1, TSP-2, and patient response to the drug. Conclusions: ABT 510, at subcutaneous doses up to 200 mg/day, is tolerated well with concurrent TMZ and RT in patients with newly diagnosed GBM. No significant financial relationships to disclose.

Angiogenic biomaker study in osteosarcoma

e21507 Background: This study represents a prospective assessment of angiogenesis genes mRNA expression in tumors and blood from patients treated with pre- and post-operative Brazilian osteosarcoma protocol (GCBTO 2006) that introduce metronomic chemotherapy (anti-angiogenic) in order to try to increase survival of osteosarcoma patients. Methods: Tumor samples from 27 patients were analyzed before and after chemotherapy to determine VEGFA, VEGFR1, VEGFR2, PDGFC, SDF1 and TSP1 genes expression profile by Quantitative Real Time PCR. Also, blood samples of these patients were investigated pre- and post-chemotherapy and at the end of high-dose chemotherapy trying to evaluate potential for proangiogenic factors and antiangiogenic factor (TSP1) which could be used to monitor treatment activity. Results: Of all six genes studied pre- and post- chemotherapy, in tumor samples, only SDF1 and VEGFR2 were underexpressed. SDF1 gene has the lowest expression at all. In tumor samples, TSP1 and VEGFA expression was higher than SDF1, VEGFR2 and PDGFC expression in biopsies and surgeries (P=0.001). VEGFR1 expression was higher than VEGFR2 expression (P=0.001). PDGFC and VEGFR1 overexpression were associated with necrosis grade I and II (Huvos score) (P=0.005). VEGFA and TSP1 were overexpressed in 96% and 92% of surgery samples, respectively. In blood samples from biopsy, surgery and end of treatment there were no statistically significant changes in the marker genes expression. Conclusions: The study suggests an association between PDGFC and VEGFR1 overexpression and lower grade necrosis. TSP1 and VEGFA were the most expressed genes in all tumor samples but TSP1 was lower than VEGFA in biopsies and VEGFA was lower than TSP1 in surgery (P=0.001). Although VEGFR2 is the primary receptor of VEGF, VEGFR1 was the most expressed VEGF receptor. No significant financial relationships to disclose.

Thrombospondin-1 modulates VEGF-A-mediated Akt signaling and capillary survival in the developing retina

Microvascular development is often perceived to result from a balance of positive and negative factors that impact signaling for proliferation and survival. The survival signaling that results from hypoxia-induced VEGF-A has been well established, but the factors that antagonize this signaling have been poorly studied. As endogenous inhibitors of angiogenesis, thrombospondins (TSPs) are likely candidates to affect survival signaling. Here we report that TSP1 antagonized microvascular survival to retinal hyperoxia, and Akt signaling in both the retina and in cultured endothelial cells. TSP1 expression is correlated with the association of the CD36 receptor with Src versus Fyn. In the presence of TSP1, CD36 is coprecipitated with Fyn as previously shown by others. However, in the absence of TSP1, there is a preferential association with Src. We now demonstrate that these Src family kinases play an important role in modulating microvascular survival in response to TSP1 by crossing tsp1(-/-) mice to the src(-/-) and fyn(-/-) mice and testing the survival of retinal blood vessels in hyperoxia. We find that tsp1(-/-), fyn(-/-), and double-mutant tsp1(-/-)/fyn(-/-) mice have a similar enhancement of capillary survival in oxygen, whereas in a tsp(-/-) background, the loss of only one allele of src restores the balance in survival and apoptosis to that of wild-type mice. Taken together, we hypothesize that TSP1 antagonizes VEGF-driven Akt survival signaling in part through the recruitment of Fyn to membrane domains containing CD36, but when TSP1 is absent, an opposing Src recruitment contributes to VEGF-driven Akt phosphorylation and capillary survival.

ORIGINAL ARTICLE: The Role of TSP‐1 on Decidual Macrophages Involved in the Susceptibility to Unexplained Recurrent Spontaneous Abortion

Problem To investigate the role of TSP‐1 on decidual macrophages (DMΦ) in unexplained recurrent spontaneous abortion (RSA).Method of study A total of 20 women undergoing artificial abortion in the first trimester and 10 patients with RSA were selected. (i) The expression of TSP‐1 mRNA in deciduas was detected by real‐time polymerase chain reaction; (ii) Flow cytometry was used to detect the percentage of positive TSP‐1, CD36, CD47 markers on macrophages. (iii) Cytokine expression was measured by enzyme‐linked immunosorbent spot‐forming (ELISPOT) cell assay.Results (i) The expression of TSP‐1 mRNA on DMΦ in RSA was significantly decreased (P &lt; 0.05). (ii) The increased expression of CD36 (P &lt; 0.01) and the decreased expression of TSP1 (P &lt; 0.01) were found on DMΦ of RSA. No different CD47 expression level on DMΦ was observed between two groups. (iii)The expression of IL‐10 in DMΦ of RSA patients was decreased significantly compared with that of controls (P &lt; 0.05). When adding TSP1 into culture medium, there was no change in IFN‐γ expression, but increased IL‐10 (P &lt; 0.05) expression in DMΦ of RSA patients was observed. The expression of IL‐10 was decreased significantly in DMΦ from controls when adding anti‐TSP‐1 antibody to culture medium (P &lt; 0.05).Conclusion TSP‐1 on DMΦ could influence IL‐10 expression as Th2 cytokines. The abnormal expression of TSP‐1 could make some women undergo pregnancy loss.

NF-κB blockade upregulates Bax, TSP-1, and TSP-2 expression in rat granulation tissue

Several diseases are characterized by chronic inflammation, a condition frequently associated with angiogenesis and fibrogenesis that account for the development of granulation tissue. Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) is a crucial modulator of intracellular prosurvival signaling pathways and is implicated in the pathogenesis of inflammatory process. In this study, we have investigated the role of NF-kappaB in the angiogenic and fibrogenic response induced by lambda-carrageenin in a rat model of chronic inflammation at 1, 3, and 5 days. The subcutaneous implant of lambda-carrageenin-soaked sponges in rat induced a time-related increase of granulation tissue formation accompanied by intense neovascularization. These lambda-carrageenin-induced changes were significantly reduced by coinjection of wild-type oligodeoxynucleotide (WT ODN) decoy to NF-kappaB. Molecular, morphological, and ultrastructural analysis performed on whole granulation tissue demonstrated: (1) inhibition of NF-kappaB/DNA binding activity; (2) downregulation of cyclooxygenase-2, matrix metalloproteinase-9, tumor necrosis factor-alpha, and vascular endothelial growth factor; (3) upregulation of thrombospondin (TSP)-1 at 1 day and TSP-2 at 5 days; and (4) increase in Bax to Bcl-2 ratio. Our findings show that the blockade of NF-kappaB activation by WT ODN decoy prevents the development of granulation tissue induced by lambda-carrageenin-soaked sponge implant upregulating Bax as well as TSP-1 and TSP-2 expression.

Markers of angiogenesis in high-risk, early-stage cervical cancer: A Gynecologic Oncology Group study

Objectives To determine whether markers of tumor angiogenesis were associated with progression-free survival (PFS) and overall survival (OS) in women with high-risk, early-stage cervical cancer treated on a phase III trial. Methods One hundred seventy-three tumor specimens were analyzed by semi-quantitative immunohistochemical (IHC) staining for vascular endothelial growth factor (VEGF, pro-angiogenesis factor), thrombospondin-1 (TSP-1, anti-angiogenesis factor), CD31 (non-specific endothelial marker), and CD105 (tumor-specific endothelial marker). Tumoral histoscores (HS) were calculated for VEGF using the formula: [% cells positive × (intensity + 1)]. TSP-1 specimens were categorized as negative or positive. CD31 and CD105 microvessel density (MVD) “hotspots” were counted in three 20× high-power fields. Associations between angiogenesis markers and survival were evaluated. Results TSP-1 expression was observed in 65% of cases while 66% expressed high VEGF (≥ 200), 34% exhibited high CD31 (CD31 ≥ 110) and 66% displayed high CD105 (CD105 ≥ 28). In univariate analyses CD31 MVD, but not tumor TSP-1, was associated with improved PFS (HR = 0.37; 95% CI = 0.18–0.76; p = 0.007) and OS (HR = 0.37; 95% CI = 0.17–0.79; p = 0.010). After adjusting for prognostic clinical covariates, high CD31 MVD, but not TSP-1, VEGF or CD105 MVD, was an independent prognostic factor for PFS (HR = 0.36; 95% CI = 0.17–0.75; p = 0.006) and OS (HR = 0.36; 95% CI = 0.17–0.79; p = 0.010). Conclusions Tumor angiogenesis measured by CD31 MVD is an independent prognostic factor for both PFS and OS in high-risk, early-stage cervical cancer. We hypothesize that this finding may be explained by improved treatment response in well-vascularized, well-oxygenated tumors.

Thrombospondin 2-null mice display an altered brain foreign body response to polyvinyl alcohol sponge implants

Thrombospondin (TSP)-2 is a matricellular protein that participates in the processes of tissue repair and the foreign body response. In addition, TSP2 has been shown to influence synaptogenesis and recovery of the brain following stroke. In the present study we investigated the response following the implantation of polyvinyl alcohol (PVA) sponges in the brain. PVA sponges were implanted into the brain cortex of wild type and TSP2-null mice for a period of 4 and 8 weeks and the response was analyzed by histochemistry and quantitative immunohistochemistry. TSP2 expression was detected in the interstices of the sponge and co-localized with the extracellular matrix and astrocytes. PVA sponge invasion in TSP2-null mice was characterized by dense deposition of extracellular matrix and increased invasion of reactive astrocytes and macrophages/microglia. Furthermore, the angiogenic response was elevated and the detection of mouse serum albumin (MSA) in the brain cortex indicated excessive vessel leakage, suggesting that TSP2 plays a role in the repair/maintenance of the blood brain barrier. Finally, immunostaining demonstrated an increase in the levels of matrix metalloproteinase (MMP)-2 and MMP-9. Taken together, our observations support a role for TSP2 as critical determinant of the brain response to biomaterials.

Abstract 310: Thrombospondin 2 Protects Against Uncontrolled Cardiac Inflammation and Injury During Viral Myocarditis

Background-The matricellular protein thrombospondin 2 (TSP2), a secreted extracellular matrix (ECM) glycoprotein, plays a crucial role in controlling cell-matrix interaction in tissue repair and cancer biology. Our previous data revealed that increased TSP2 reinforces the matrix and protects the against heart failure during hypertension. The present study investigated whether TSP2 may protects against exaggerated cardiac inflammation, injury and dysfunction during viral myocarditis. Methods and Results-Therefore, mortality, cardiac inflammation and function were investigated in TSP2-null (KO) and wild type (WT) mice, and after adeno-associated virus (AAV)-9 mediated TSP-2 overexpression in WT, in a mouse model of coxsackievirus B3 (CVB3)-induced (106 CCID50 ip CBV3, 14 days) myocarditis. Absence of TSP2 resulted in an increased mortality in TSP2-null as compared with WT mice in both viral myocarditis (KO: 37,5% vs WT: 0%; n=8 per group). Histological analysis revealed increased cardiac inflammation (60 % increase of CD3-positive T-lymphocytes), necrosis and injury in TSP2-null compared to WT mice at 14 days of viral myocarditis. Ultrastructural analysis showed increased inflammation, matrix disruption and myocyte injury in absence of TSP2, together resulting in depressed systolic function (% FS; 40.1 ± 2.3 in WT vs. 24.6 ± 1.7 in KO mice, P&lt;0.05) and increased cardiac dilatation (end-diastolic dimensions, mm; 2.6 ± 0.18 in WT vs. 3.6 ± 0.06 in KO mice, P&lt;0.05) at 14 days at echocardiography. In concordance, AAV9-gene transfer of TSP2, with 50 % increase in cardiac TSP-2 expression, significantly reduced cardiac inflammation (60% reduction) and injury at 14 days as compared to control AAV9-GFP. Conclusion-Together, these data clearly point towards a novel protective role for TSP2 against uncontrolled inflammation, myocardial injury and dysfunction in acute viral myocarditis.

Thrombospondin 1 Promotes Tumor Macrophage Recruitment and Enhances Tumor Cell Cytotoxicity of Differentiated U937 Cells

Inhibition of tumor growth by thrombospondin (TSP) 1 is generally attributed to its antiangiogenic activity, but effects on tumor immunity should also be considered. We show that overexpression of TSP1 in melanoma cells increases macrophage recruitment into xenograft tumors grown in nude or beige/nude mice. In vitro, TSP1 acutely induces expression of plasminogen activator inhibitor-1 (PAI-1) by monocytic cells, suggesting that TSP1-induced macrophage recruitment is at least partially mediated by PAI-1. Tumor-associated macrophages (TAM) can either promote or limit tumor progression. The percentage of M1-polarized macrophages expressing inducible nitric oxide synthase is increased in TSP1-expressing tumors. Furthermore, soluble TSP1 stimulates killing of breast carcinoma and melanoma cells by IFN-gamma-differentiated U937 cells in vitro via release of reactive oxygen species. TSP1 causes a significant increase in phorbol ester-mediated superoxide generation from differentiated monocytes by interaction with alpha(6)beta(1) integrin through its NH(2)-terminal region. The NH(2)-terminal domain of TSP2 also stimulates monocyte superoxide production. Extracellular calcium is required for the TSP1-induced macrophage respiratory burst. Thus, TSP1 may play an important role in antitumor immunity by enhancing recruitment and activation of M1 TAMs, which provides an additional selective pressure for loss of TSP1 and TSP2 expression during tumor progression.

PDE-5 inhibition impedes TSP-1 expression, TGF- activation and matrix accumulation in experimental glomerulonephritis

Matrix expansion and mesangial proliferation are hallmarks of mesangial proliferative glomerulonephritis. Specific inhibition of PDE-5, an enzyme catalyzing the intracellular degradation of cyclic GMP, can be achieved by the inhibitor vardenafil. In this study, we investigated the effects of PDE-5 inhibition in the anti-Thy1 model in the rat in vivo. After disease induction, rats received 10 mg/kg bw vardenafil twice a day via gavage. On Days 2 and 6, renal biopsies, as well as glomerular isolates, urine and blood samples were taken to compare vardenafil- and placebo-treated groups during the course of disease. Small amounts of PDE-5 were detected in healthy kidneys, but induced in a typical mesangial pattern during disease (by IHC and WB). Specific PDE-5 inhibition resulted in increased glomerular levels of cGMP. Treated animals demonstrated inhibition of MC proliferation and matrix accumulation while renal function and influx of inflammatory cells were not affected. Due to PDE-5 inhibition, the endogenous TGF-beta-activating protein TSP-1 and the TGF-beta-signalling protein p-smad-2/3 were decreased suggesting this as an antifibrotic mechanism of action of vardenafil in this model. Considering the availability and safety profile of vardenafil, the beneficial antiproliferative and antifibrotic effect in experimental glomerulonephritis may potentially be applicable to the treatment of mesangial proliferative glomerulonephritis in man.

Down regulation of c-Myc and induction of an angiogenesis inhibitor, thrombospondin-1, by 5-FU in human colon cancer KM12C cells

5-FU is commonly used for treatment of various solid tumors including colon carcinoma. We have previously demonstrated that Egr-1 induced by 5-FU enhanced TSP-1 expression in human colon cancer KM12C cells. In this study, a Genechip analysis of KM12C cells treated with 5-FU revealed down-regulation of 924 genes and up-regulation of 460 genes. The decreased expression of c-Myc mRNA and phosphorylated c-Myc were detected and confirmed by RT-PCR and immunoblotting. Since 5-FU induced the expression of TSP-1, we examined the effect of c-Myc on the TSP-1 promoter. Deletion of the TSP-1 promoter region in which binding sites for c-Myc reside had no effect on the TSP-1 promoter activity induced by 5-FU. Meanwhile, 5-FU dose-dependently decreased the expression of miR-17-92 cluster. These findings suggest that 5-FU decreased the expression of c-Myc and consequently miR-17-92 cluster and increased the expression of TSP-1 mRNA.

Thrombospondin-4 expression is rapidly upregulated by cardiac overload

The precise mechanisms regulating gene expression of thrombospondins (TSPs) in the heart remain incompletely understood. Here we characterized cardiac TSP-4 expression in response to pressure overload and myocardial infarction in vivo. Arginine 8-vasopressin (AVP) infusion increased left ventricular (LV) TSP-4 mRNA levels within 30 min. Also angiotensin II infusion rapidly activated LV TSP-4 expression, TSP-4 mRNA levels being highest at 6 h and protein at 72 h and 2 weeks. During remodeling process following myocardial infarction, LV TSP-4 mRNA levels increased at day one, as studied by quantitative RT-PCR. TSP-4 immunostaining was localized to endothelial cells in hypertrophied hearts of spontaneously hypertensive rats. AVP-infusion increased LV TSP-1 mRNA levels similarly to TSP-4 within 30 min showing that rapid induction of gene expression, well before the development of cardiac hypertrophy, is typical for the thrombospondin family. These results further suggest that TSP-4 may be an endothelial specific marker of cardiac overload.

Thrombospondin-2 inhibits tumor cell invasion through the modulation of MMP-9 and uPA in pancreatic cancer cells

The extracellular matrix protein thrombospondin (TSP) plays an important role in a variety of biological processes, including cell-cell and cell-matrix interactions. The biological role of TSP-2 in invasion and metastasis is poorly understood, while it is known that TSP-1 regulates a proteolytic cascade that allows tumor cells to invade and metastasize. In this study, we examined the role of TSP-2 in tumor cell invasion and its association with proteolytic proteins, matrix metalloproteinase (MMP) and the plasminogen/plasmin system, including urokinase-type plasminogen activator (uPA), in the human pancreatic cancer cell line PANC-1. PANC-1 cells expressed a low level of TSP-2, but significant levels of TSP-1. We isolated three clones of PANC-1 transformants stably overexpressing human TSP-2 (PANC-T). PANC-T highly expressed the TSP-2 gene and protein, while TSP-1 expression was not altered. In vitro invasion assays demonstrated that the invasiveness of PANC-T clones was significantly suppressed (p<0.05; Welch test). Zymography revealed that restoration of TSP-2 synthesis in the PANC-T clones significantly inhibited MMP-9 activity (p<0.05; Welch test). uPA activity in the PANC-T clones was significantly suppressed (p<0.05; Welch test). We concluded that restoration of TSP-2 can inhibit cell invasion through the down-regulation of MMP-9 and uPA activity in pancreatic cancer cell lines. Thus, TSP-2 may be a potent inhibitor of metastasis in pancreatic cancer.

Antiangiogenic and anticolorectal cancer effects of metronomic irinotecan chemotherapy alone and in combination with semaxinib

Metronomic chemotherapy refers to the administration of chemotherapy at low, nontoxic doses on a frequent schedule with no prolonged breaks. The aim of the study is to rationally develop a CPT-11 metronomic regimen in preclinical settings of colon cancer. In vitro cell proliferation, apoptosis and thrombospondin-1/vascular endothelial growth factor (TSP-1/VEGF) expression analyses were performed on endothelial (HUVEC, HMVEC-d) and colorectal cancer (HT-29, SW620) cells exposed for 144 h to metronomic concentrations of SN-38, the active metabolite of CPT-11. HT-29 human colorectal cancer xenograft model was used, and tumour growth, microvessel density and VEGF/TSP-1 quantification was performed in tumours. In vitro and in vivo combination studies with the tyrosine inhibitor semaxinib were also performed. SN-38 preferentially inhibited endothelial cell proliferation alone and interacted synergistically with semaxinib; it induced apoptosis and increased the expression and secretion of TSP-1. Metronomic CPT-11 alone and combined with semaxinib significantly inhibits tumour growth in the absence of toxicity, which was accompanied by decreases in microvessel density and increases in TSP-1 gene expression in tumour tissues. In vitro results show the antiangiogenic properties of low-concentration SN-38, suggesting a key role of TSP-1 in this effect. In vivo, the CPT-11 metronomic schedule is effective against tumour and microvessel growth without toxic effect on mice.