Thrombospondin-1 Expression Research Articles

Tsp1 promotes alveolar stem cell proliferation and its down-regulation relates to lung inflammation in intralobar pulmonary sequestration.

An aberrant systemic artery supply results in recurrent infections in the abnormal lung lobe of intralobar pulmonary sequestration (ILS). The mechanisms underlying such persistent inflammation are unknown. Here, we hypothesize that alteration of an endothelial cell niche for alveolar epithelial cells results in the impaired proliferation potential of alveolar progenitor cells, leading to the defective defense mechanism in intralobar pulmonary sequestration. Paraffin sections of lung tissues from patients with intralobar pulmonary sequestration or from healthy controls were collected for analysis of alveolar epithelial alterations in intralobar pulmonary sequestration by quantitative RT-PCR or immunofluorescent staining. Differential transcripts were identified between human pulmonary artery endothelial cells and human aortic endothelial cells by microarray. Validation of microarray data by quantitative PCR analysis indicated that thrombospondin-1 expression level is low in near-lesion part but high in lesion part of ILS lobe as compared to healthy controls. In vitro 3-D matrigel culture was adopted to evaluate the regulation of alveolar progenitor cells by thrombospondin-1 and CD36. We found that the proliferative potential of alveolar type 2 stem/progenitor cells was impaired in intralobar pulmonary sequestration. Mechanistically, we discovered that endothelial thrombospondin-1 promotes alveolar type 2 cell proliferation through the interaction with CD36. These data demonstrate that alveolar stem cells are impaired in the abnormal lobe from patients with intralobar pulmonary sequestration and imply that restoring epithelial integrity can be beneficial for the future treatments of recurrent infections in lung pathologies.

Renal Protection Mediated by Hypoxia Inducible Factor-1α Depends on Proangiogenesis Function of miR-21 by Targeting Thrombospondin 1

BackgroundAngiogenesis contributes to the repair process after renal ischemia/reperfusion (I/R) injury. In the present study, we tested the role of miR-21 in the angiogenesis induced by hypoxia inducible factor (HIF)-1α through inhibiting a predicted target gene thrombospondin 1 (TSP-1).MethodsTo stabilize HIF-1α, hypoxia (1% O2 for 24 hours) was performed in human umbilical vein endothelial cells and cobalt chloride (CoCl2) was pretreated intraperitoneally 24 hours before renal I/R in mice. Locked nucleic acid modified anti-miR-21 and scrambled control was transfected with hypoxic cells or delivered into the mice via tail vein 1 hour before CoCl2 injection. The kidneys and blood were collected at 24 hours after reperfusion.ResultsHIF-1α induced by hypoxia and CoCl2 upregulated vascular endothelial growth factor and miR-21, and increased angiogenesis. It was found that expression of TSP-1 was inversely related with miR-21 in vitro and in vivo. Targeting of TSP-1 by miR-21 was further confirmed in vitro. Furthermore, HIF-1α improved renal function, accompanied with increased angiogenesis after I/R injury in mice. The protective effect of HIF-1α was attenuated by inhibition of miR-21.ConclusionsHIF-1α induced angiogenesis by upregulating not only vascular endothelial growth factor but also miR-21 via inhibiting a novel target gene TSP-1. Both of them may contribute to the protective effect of HIF-1α on renal I/R injury.

15-Lipoxygenase-1 re-expression in colorectal cancer alters endothelial cell features through enhanced expression of TSP-1 and ICAM-1

15-lipoxygenase-1 (15-LOX-1) oxygenates linoleic acid to 13(S)-hydroxyoctadecadienoic acid (HODE). The enzyme is widely suppressed in different cancers and its re-expression has tumor suppressive effects. 15-LOX-1 has been shown to inhibit neoangiogenesis in colorectal cancer (CRC); in the present study we confirm this phenomenon and describe the mechanistic basis. We show that re-expression of 15-LOX-1 in CRC cell lines resulted in decreased transcriptional activity of HIF1α and reduced the expression and secretion of VEGF in both normoxic and hypoxic conditions. Conditioned medium (CM) was obtained from CRC or prostate cancer cell lines re-expressing 15-LOX-1 (15-LOX-1CM). 15-LOX-1CM treated aortic rings from 6-week old C57BL/6 mice showed significantly less vessel sprouting and more organized structure of vascular network. Human umbilical vein endothelial cells (HUVECs) incubated with 15-LOX-1CM showed reduced motility, enhanced expression of intercellular cell adhesion molecule (ICAM-1) and reduced tube formation but no change in proliferation or cell-cycle distribution. HUVECs incubated with 13(S)-HODE partially phenocopied the effects of 15-LOX-1CM, i.e., showed reduced motility and enhanced expression of ICAM-1, but did not reduce tube formation, implying the importance of additional factors. Therefore, a Proteome Profiler Angiogenesis Array was carried out, which showed that Thrombospondin-1 (TSP-1), a matrix glycoprotein known to strongly inhibit neovascularization, was expressed significantly more in HUVECs incubated with 15-LOX-1CM. TSP-1 blockage in HUVECs reduced the expression of ICAM-1 and enhanced cell motility, thereby providing a mechanism for reduced angiogenesis. The anti-angiogenic effects of 15-LOX-1 through enhanced expressions of ICAM-1 and TSP-1 are novel findings and should be explored further to develop therapeutic options.

T-cell regulation through a basic suppressive mechanism targeting low-density lipoprotein receptor-related protein 1.

Cell adhesion is generally considered to depend on positive regulation through ligation of integrins and cytokine receptors. However, here we show that T-cell adhesion, and notably also T-cell receptor (TCR) -induced activation, are subject to constant suppression through shedding of low-density lipoprotein receptor-related protein 1 (LRP1). The broad-spectrum metalloprotease inhibitor GM6001 abrogated shedding, so inducing prominent cell surface expression of LRP1 while enhancing TCR-induced activation and adhesion to β1 and β2 integrin ligands, hence arresting the cells. Integrin ligands also inhibited shedding but the effect was less potent than that of GM6001. Unlike GM6001, integrin ligands also induced cell surface expression of full-length thrombospondin-1 (TSP170) and TSP130, which associated with LRP1, and TSP110, which did not associate with LRP1. Cell surface expression of LRP1 and TSP130 were induced exclusively in adhering cells, expression of TSP110 preferentially in non-adhering cells and expression of TSP170 correlated with T-cell motility. The pro-adhesive chemokine CXCL12 also inhibited LRP1 shedding and induced surface expression of TSP170 and TSP130 while inhibiting TSP110. Exogenous TSP-1 and ligation of CD28 inhibited shedding although less effectively than GM6001, and the inhibition through CD28 was independent of TSP-1. Small interfering RNA silencing experiments confirmed involvement of LRP1 and TSP-1 in integrin-dependent adhesion and TCR-induced activation. Hence, the poor LRP1 expression in T cells depends on shedding. Integrin ligands and CXCL12 antagonize shedding through a TSP-1-dependent pathway and ligation of CD28 antagonizes shedding independent of TSP-1. The disappearance of LRP1 from the cell surface may provide basic immunosuppression at the T-cell level.

Correlation Between Thrombospondin-1 Expression in Non-cancer Tissue and Gastric Carcinogenesis.

Thrombospondin-1 (TSP1) is correlated with carcinogenesis occurring in cases of intestinal inflammation. The aim of this study was to clarify the role of TSP1 in gastric carcinogenesis. A total of 39 patients with gastric cancer who had undergone gastrectomy were enrolled. The expression of TSP1 mRNA in non-cancer tissues was determined. Furthermore, the expression of CD36, STAT3 and TGFβR2 mRNA in non-cancer tissues in two expression groups, the TSP1 high- and low-expression groups, were examined. The expression of TSP1 was high in the mucosal-atrophy group and tended to be high in the Helicobacter pylori (H. pylori) (+) and multiple cancer groups. The levels of CD36, STAT3 and TGFβR2 mRNA were significantly higher in the TSP1-high group. TSP1 signaling pathway was induced in multiple cancer or atrophy (+) or H. pylori (+) compared to cases with single cancer, atrophy (-) and H. pylori (-). Expression of proteins involved in the TSP1 signaling pathway in non-cancer tissues with multiple gastric cancers were higher than that with single gastric cancer. Expression of TSP1 in non-cancer tissue correlated with gastric carcinogenesis.

Abstract LB-213: Thrombospondin-1 regulates intestinal microbiota and bile acid metabolism in a murine model of colorectal cancer

Abstract Colon cancer is major public health concern with over 50,000 deaths annually in the US. Environmental and genetic factors influence colon cancer progression, and expression of thrombospondin-1 (TSP1) inversely correlates with colon cancer aggressiveness. TSP1 is a matricellular protein that regulates vascular physiology and tissue responses to stress. We previously demonstrated that deleting the TSP1 (Thbs1) gene in ApcMin/+ mice results in systemic metabolic changes as assessed in liver tissue, increased tumorigenesis, and lower survival of mice fed a low fat diet. We now show that TSP1 induces changes in intestinal microbiota that may cause shifts in secondary bile acid metabolism that influence colon carcinogenesis. Mice were maintained and bred at least two generations on a low fat western diet to equalize dietary effects on their epigenetic context and microbiomes. Mice were pair-fed a low fat or a 21% high-fat western diet beginning at weaning. Mouse fecal matter was collected 8 weeks after dietary exposures and subjected to 16S sequencing for bacterial population identification. Lactobacillaceae, Lachnopiraceae, Ruminococcacea, Verrucomicrobiaceae families were present in all groups. The family Verrucomicrobiaceae and its genus Akkemensi represented a high proportion of the bacterial population. The relative levels of Akkemensia were elevated in low fat fed mice in mice ApcMin/+ and Thbs1−/−:ApcMin/+ when compared to WT counterparts. Interestingly Akkenmensia abundance was reduced in ApcMin/+ mice fed a high-fat diet when compared to other groups. The relative population of the genus Ruminococcus was reduced in Thbs1−/− mice as well as in Thbs1−/−:ApcMin/+ mice fed low and high fat diets, suggesting that these population shifts are driven by genotype. Because intestinal bacteria can alter metabolism, particularly of bile acids, we collected large intestine tissue from mice and subjected it to metabolomics analysis. Our data showed primary and secondary bile metabolism were regulated by the absence of TSP1 in mice fed a high fat diet when compared to WT. High 6-beta-hydroxylithocholate levels were associated with onset of carcinogenesis since it was elevated in ApcMin/+ and Thbs1−/−:ApcMin/+ mice but not in WT or Thbs1−/−. 6-oxolithocholate, hyocholate and hyodeoxycholate were elevated only in ApcMin/+ mice fed a high fat diet but not in Thbs1−/−:ApcMin/+, suggesting that these metabolites contribute to the shift in survival and carcinogenesis observed between low and high fat diets. On the other hand levels of taurohyodeoxycholic acid, 3-dehydrocholate, 7-ketodeoxycholate were elevated in Thbs1−/−:ApcMin/+ and Thbs1−/− mice but not in ApcMin/+, indicating that changes in these metabolites are driven by the loss of Thbs1 and may pre-dispose mice to increased carcinogenesis when fed a high fat diet. Taken together our data shows an unexpected role of TSP1 in regulation of microbiota and bile acid metabolism and reveals potential targets for prevention and treatment of colorectal cancer. Citation Format: David R. Soto-Pantoja, John M. Sipes, Brian Westwood, Nicole Morris, Nancy J. Emenaker, David D. Roberts. Thrombospondin-1 regulates intestinal microbiota and bile acid metabolism in a murine model of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-213. doi:10.1158/1538-7445.AM2017-LB-213

Abstract 4808: Effect of Merkel cell polyomavirus large and small T-antigen on the thrombospondin and the periostin promoter

Abstract Merkel cell carcinoma (MCC) is a rare, but aggressive form of skin cancer with rising incidence and high mortality rate. Approximately 80% of MCC tumors are positive for Merkel cell polyomavirus (MCPyV), suggesting a causative relationship between MCPyV and MCC. Exosomes are 30-150 nm vesicles that contain proteins, lipids, mRNAs, lncRNAs, and miRNAs. Exosomes from virus-infected cells comprise also viral nucleic sequences and proteins. Because tumor cell-derived exosomes may contribute to cancer, we hypothesized that MCPyV may affect the composition of exosomes and play a role in tumorigenesis. Therefore, we compared the proteins in exosomes produced by MCPyV-positive MKL1 and MKL2 and MCPyV-negative MCC13 and MCC26 MCC cell lines. Our proteomic analysis of exosomes originating from polyomavirus-negative and polyomavirus-positive MCC cell lines revealed the presence of the oncogenic proteins periostin and thrombospondin. Western blot analysis of exosomes and lysates of these MCC cells confirmed the presence of these proteins in exosomes from all cell lines. Thrombospondin, but not periostin was detectable in cell lysates, and an enrichment of both proteins was detected in exosomes of all cell lines. The effect of MCPyV large T-antigen (LT-ag) and small t-antigen (st-ag) on the periostin and thrombospondin promoters was examined by transient transfection studies with luciferase reporter plasmids. Transfection experiments in MCC13 revealed that LT-ag, but not st-ag significantly increased the activity of the thrombospondin and periostin promoters. In MCC26 cells, neither LT-ag nor st-ag had a significant effect on the thrombospondin promoter activity, while both proteins alone or in combination significantly stimulated the periostin promoter strength. Our results suggest that MCPyV proteins may contribute to tumorigenesis by enhancing the expression of the oncoproteins thrombospondin and periostin and promote their secretion via exosomes. Citation Format: Aelita Konstantinell, Ida Sofie Furuholmen, Baldur Sveinbjørnsson, Ugo Moens. Effect of Merkel cell polyomavirus large and small T-antigen on the thrombospondin and the periostin promoter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4808. doi:10.1158/1538-7445.AM2017-4808

Thrombospondin-1 Production Regulates the Inflammatory Cytokine Secretion in THP-1 Cells Through NF-κB Signaling Pathway.

Thrombospondin-1 (TSP-1) is upregulated in several inflammatory diseases. Recent data have shown that macrophages from TSP-1-deficient mice have a reduced inflammatory phenotype, suggesting that TSP-1 plays a part in macrophage activation. DNA microarray approach revealed that Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) may induce the enhanced TSP-1 expression in human monocytes, suggesting a role of TSP-1-mediated pathogenesis in periodontitis. Until recently, the function of TSP-1 has been a matter of debate. In this study, we explored the role of TSP-1 in inflammatory cytokine secretions and its putative mechanism in pathogenesis of periodontitis. We demonstrated that TSP-1 expression was significantly upregulated in gingival tissues with periodontitis and in P. gingivalis LPS-stimulated THP-1 cells. Deficiency of TSP-1 by transfecting siRNAs decreased IL-6, IL-1β, and TNF-α secretions in THP-1 cells, whereas overexpression of TSP-1 resulted in an upregulation of IL-6, IL-1β, and TNF-α productions. Additional experiments showed that Pyrrolidine dithiocarbamate (PDTC) inhibited IL-6, IL-1β, and TNF-α expression induced by overexpression of TSP-1, accompanying with downregulation of phosphorylated p65 and IκBα protein levels in response to P. gingivalis LPS. These results indicated that TSP-1 played a significant role in P. gingivalis LPS-initiated inflammatory cytokines (IL-6, IL-1β, and TNF-α) secretions of THP-1 cells, and the NF-κB signaling is involved in its induction of expression. Thus, TSP-1 effectively elevated P. gingivalis LPS-induced inflammation mediated by the NF-κB pathway and may be critical for pathology of periodontitis.

Hematopoietic Id Deletion Triggers Endomyocardial Fibrotic and Vascular Defects in the Adult Heart

Inhibitor of DNA binding (Id) proteins play important roles in regulating cardiac development via paracrine signaling. Id1/Id3 knockout mice die at mid-gestation with multiple cardiac defects. Single Id knockout studies have not reported cardiomyopathies. To bypass embryonic lethality we used Tie2CRE-mediated recombination to conditionally delete Id1 against global Id3 ablation (Id cDKOs), which develops adult-onset dilated cardiomyopathy. We confirm upregulation of thrombospondin-1 (TSP1) in Id cDKO hearts. Colocalization studies reveal increased TSP1 expression in the vicinity of endothelial cells and near regions of endocardial fibrosis/disruption. Downstream fibrotic molecules were upregulated. Endocardial capillary density was reduced with evidence of vascular distention. Treatment of Id cDKO cardiac explants with LSKL, a peptide antagonist of TSP1 activation of TGFβ, reversed the increased expression of fibrotic molecules. We conducted bone marrow transplant experiments in which we transferred bone marrow cells from Id cDKO mice into lethally irradiated WT mice. The majority of WT recipients of Id cDKO bone marrow cells phenocopied Id cDKO cardiac fibrosis 4 months post-transplantation. Injection of LSKL into adult Id cDKO mice led to downregulation of fibrotic molecules. The results prompt caution when bone marrow transfers from individuals potentially carrying mutations in the Id axis are applied in clinical settings.

Aureobasidium pullulans produced \u03b2-glucan is effective to enhance Kurosengoku soybean extract induced Thrombospondin-1 expression

Black yeast, Aureobasidium pullulans is extracellularly produced β-(1,3), (1,6)-D-glucan (β-glucan) under certain conditions. In this study, using Glycine max cv. Kurosengoku (Kurosengoku soybeans), the production of β-glucan through fermentation of A. pullulans was evaluated, and the effects of A. pullulans cultured fluid (AP-CF) containing β-glucan made with Kurosengoku soybeans (kAP-CF) on a human monocyte derived cell line, Mono Mac 6 cells were investigated. Concentration of β-glucan in kAP-CF reached the same level as normal AP-CF. An anti-angiogenic protein, Thrombospondin-1 (THBS1) was effectively induced after the stimulation with kAP-CF for comparison with AP-CF. The THBS1 is also induced after stimulation with hot water extract of Kurosengoku soybeans (KS-E), while the combined stimulation of β-glucan with KS-E more effectively induced THBS1 than that with KS-E alone. These results suggest effects of A. pullulans-produced β-glucan on the enhancement of Kurosengoku soybean-induced THBS1 expression.

Induction of senescence in primary glioblastoma cells by serum and TGF\u03b2

Glioblastoma is the most common type of adult brain tumour and has a median survival after diagnosis of a little more than a year. Glioblastomas have a high frequency of mutations in the TERT promoter and CDKN2A locus that are expected to render them resistant to both replicative and oncogene-induced senescence. However, exposure of PriGO8A primary glioblastoma cells to media with 10% serum induced a senescence-like phenotype characterized by increased senescence-associated β galactosidase activity, PML bodies and p21 and morphological changes typical of senescence. Microarray expression analysis showed that 24 h serum exposure increased the expression of genes associated with the TGFβ pathway. Treatment of PriGO8A cells with TGFβ was sufficient to induce senescence in these cells. The response of PriGO8A cells to serum was dependent on basal expression of the TGFβ activator protein thrombospondin. Primary glioblastoma cells from three additional patients showed a variable ability to undergo senescence in response to serum. However all were able to undergo senescence in response to TGFβ, although for cells from one patient this required concomitant inhibition of Ras pathway signalling. Primary glioblastoma cells therefore retain a functional senescence program that is inducible by acute activation of the TGFβ signalling pathway.

Inflammatory Differences in Plaque Erosion and Rupture in Patients With ST-Segment Elevation Myocardial Infarction.

BackgroundPlaque erosion causes 30% of ST‐segment elevation myocardial infarctions, but the underlying cause is unknown. Inflammatory infiltrates are less abundant in erosion compared with rupture in autopsy studies. We hypothesized that erosion and rupture are associated with significant differences in intracoronary cytokines in vivo.Methods and ResultsForty ST‐segment elevation myocardial infarction patients with <6 hours of chest pain were classified as ruptured fibrous cap (RFC) or intact fibrous cap (IFC) using optical coherence tomography. Plasma samples from the infarct‐related artery and a peripheral artery were analyzed for expression of 102 cytokines using arrays; results were confirmed with ELISA. Thrombectomy samples were analyzed for differential mRNA expression using quantitative real‐time polymerase chain reaction. Twenty‐three lesions were classified as RFC (58%), 15 as IFC (38%), and 2 were undefined (4%). In addition, 12% (12 of 102) of cytokines were differentially expressed in both coronary and peripheral plasma. I‐TAC was preferentially expressed in RFC (significance analysis of microarrays adjusted P<0.001; ELISA IFC 10.2 versus RFC 10.8 log2 pg/mL; P=0.042). IFC was associated with preferential expression of epidermal growth factor (significance analysis of microarrays adjusted P<0.001; ELISA IFC 7.42 versus RFC 6.63 log2 pg/mL, P=0.036) and thrombospondin 1 (significance analysis of microarrays adjusted P=0.03; ELISA IFC 10.4 versus RFC 8.65 log2 ng/mL, P=0.0041). Thrombectomy mRNA showed elevated I‐TAC in RFC (P=0.0007) epidermal growth factor expression in IFC (P=0.0264) but no differences in expression of thrombospondin 1.ConclusionsThese results demonstrate differential intracoronary cytokine expression in RFC and IFC. Elevated thrombospondin 1 and epidermal growth factor may play an etiological role in erosion.

Expression of thrombospondin-1 and CD36 and CD47 receptors in the rat brain after exposure to damaging factors in the early postnatal period

The expression of thrombospondin-1 (TS-1) and its receptors CD47 and CD36 in the cerebral cortex and hippocampus of rats under damaging factors in the early postnatal period was studied. After hypoxia on the 7th day of postnatal development, an increase in the number of CD47-expressing cerebral endothelial cells (days of postnatal development: P28–P70) and reduction in the number of TS-1-expressing astrocytes in the cortex at P28 were observed. In animals subjected to early postnatal stress at the age of P2–P15, a decrease in TS-1-expressing astrocytes in the cortex and hippocampus was registered (predominantly at the age of P28). It was noted that these changes characterize the period of long-term effects (P28–P70) of early stress that is relevant to the processes of reparative angiogenesis and arresting of neurological deficits.

Abstract 428: Role of Thrombospondin-1 in Atherosclerotic Complications Associated With Metabolic Syndrome

Atherosclerosis is the leading cause of increased morbidity and mortality in metabolic syndrome (MetS), a constellation of risk factors that include hyperglycemia and visceral obesity. Individuals with MetS have increased incidence of vascular smooth muscle cell (VSMC) migration and proliferation, a key characteristic of VSMC phenotypic transition. We previously reported that high glucose and high leptin, mimicking hyperglycemia and obesity, independently upregulate a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), expression in VSMCs. The goal of the present study was to investigate the role of TSP-1 in development of atherosclerosis in MetS. We generated a mouse model of combined MetS and atherosclerosis ( KKAy +/- /ApoE -/- ) by crossing obese hyperglycemic agouti yellow KKAy +/- mice with atherosclerotic ApoE -/- mice. Upon weaning, yellow KKAy +/- /ApoE -/- mice and age-matched black KKAy -/- /ApoE -/- littermates were maintained on regular chow diet from 4-18 wks of age. Mice were euthanized after an overnight fasting; blood, aortae and heart were collected for lipid profiling, immunoblotting and atherosclerotic lesion studies. Yellow KKAy +/- /ApoE -/- mice showed significant increase in body weight, blood glucose and plasma lipid levels vs. black KKAy -/- /ApoE -/- littermates. Aortic root morphometry demonstrated increased lesion burden (Oil red O) and neointimal thickening (H &amp; E) in MetS KKAy +/- /ApoE -/- vs. non-MetS KKAy -/- /ApoE -/- mice. Immunohistochemistry further revealed co-localization of α-SMA (SMC marker) and PCNA (proliferation marker) expression in aortic roots of KKAy +/- /ApoE -/- vs. KKAy -/- /ApoE -/- mice. Notably, lesion formation was associated with an increase in TSP-1 expression in aortic vessels. Consistently, TSP-1 and PCNA expression were elevated in aortic SMC (aSMC) primary cultures derived from obese diabetic KKAy +/- mice vs. lean non-diabetic KKAy -/- littermates. Finally, incubation of aSMC with a TSP-1 blocking peptide reduced PCNA and vimentin (SMC synthetic marker) expression coupled with attenuated SMC proliferation in obese diabetic KKAy +/- mice. Together, these results suggest a putative role of TSP-1 in accelerated atherosclerosis and VSMC phenotypic transition in MetS.

Thrombospondin-1 promotes cell migration, invasion and lung metastasis of osteosarcoma through FAK dependent pathway.

Microenvironment at the metastatic locus usually differs greatly from that present in the site of primary tumor formation and it has a significant impact on the fate of the extravasated cancer cells. We compared gene expression signatures of primary tumors and lung metastatic tumors, and identified Thrombospondin-1 (TSP1) as highly up-regulated in the lung metastatic tumors. Immunohistochemical staining further indicated that TSP1 protein expression was higher in lung metastatic tumors compared to primary tumors in both osteosarcoma xenograft model and human clinical samples. TSP1 mRNA level is significantly associated with the Enneking stage of osteosarcoma and lung metastasis. TGF-β pathways could stimulate the TSP1 expression in osteosarcoma cells. Knockdown of TSP1 expression in osteosarcoma cells dramatically suppressed cell wound healing, migration and invasion. Treatment with recombinant TSP1 protein in osteosarcoma cells significantly promoted cell wound healing, migration and invasion. Meanwhile, suppression of TSP1 in osteosarcoma cells resulted in decreased pulmonary metastasis in vivo. Mechanistically, TSP1 increased expression of metastasis related genes, including MMP2, MMP9 and Fibronectin 1. TSP1 promoted osteosarcoma cell motility through the activation of FAK pathway. Taken together, our study provides evidence of the contributions of TSP1 to the lung metastasis of osteosarcoma and suggests that this protein may represent a potential therapeutic target for osteosarcoma lung metastasis.

MP13-20 THROMBOSPONDIN-1 HAS A POSSIBILITY OF BIOMARKER PREDICTING THE PROGRESSION OF BENIGN PROSTATIC HYPERPLASIA.

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Epidemiology & Evaluation1 Apr 2017MP13-20 THROMBOSPONDIN-1 HAS A POSSIBILITY OF BIOMARKER PREDICTING THE PROGRESSION OF BENIGN PROSTATIC HYPERPLASIA. Takashi Hamakawa, Shoichi Sasaki, Yuya Ota, Naoko Unno, Rika Banno, Masa Takada, Yasue Kubota, Kenjiro Kohri, and Takahiro Yasui Takashi HamakawaTakashi Hamakawa More articles by this author , Shoichi SasakiShoichi Sasaki More articles by this author , Yuya OtaYuya Ota More articles by this author , Naoko UnnoNaoko Unno More articles by this author , Rika BannoRika Banno More articles by this author , Masa TakadaMasa Takada More articles by this author , Yasue KubotaYasue Kubota More articles by this author , Kenjiro KohriKenjiro Kohri More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.463AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Many factors affect the pathogenesis of benign prostatic hyperplasia (BPH). Previously, we reported that interleukin-18 (IL-18) may promote stromal hyperplasia in the prostate by inducing production of thrombospondin-1 (TSP-1), as known a regulator of angiogenesis and an activator of latent transforming growth factor beta, using BPH rat model and human cultured prostatic cells. Therefore, in this study, we aimed to determine the expression levels of IL-18 and TSP-1 in human prostate tissue and assess the roles of these expressions as biomarkers to diagnose the progression of BPH. METHODS Study 1: We enrolled 28 patients without malignancy who underwent transperineal prostate biopsy at our institution. We obtained prostate tissues from the transitional zone and used these samples for total RNA extraction and cDNA preparation. The expression levels of IL-18 and TSP-1 mRNAs were evaluated by quantitative real-time reverse transcription polymerase chain reaction. We evaluated the correlation between mRNA expression levels and age, total prostate volume (TPV), transitional zone volume (TZV), transitional zone index (TZI), and serum PSA levels using Pearson's product-moment correlation coefficient. Study 2: Samples from 11 of the patients in Study 1 were used to measure the volume of the prostate at biopsy and at a given point in time after biopsy. We evaluated the correlation between the increase in prostate volume per month and mRNA expression. RESULTS Study 1: There were no correlations among age, serum PSA levels, and mRNA expression. TSP-1 expression was positively correlated with TPV (r = 0.696) and TZV (r = 0.629). Study 2: The mean observation period was 23.9 ± 13.4 months, and the mean increase in prostate volume was 10.4 ± 12.3 ml. There was a strong positive correlation between the increase in volume per month and TSP-1 expression (r = 0.663). CONCLUSIONS TSP-1 expression was positively correlated with prostate volume and the increase in prostate volume per month. This result suggested that TSP-1 might be a potential biomarker for predicting the development of BPH. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e161 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Takashi Hamakawa More articles by this author Shoichi Sasaki More articles by this author Yuya Ota More articles by this author Naoko Unno More articles by this author Rika Banno More articles by this author Masa Takada More articles by this author Yasue Kubota More articles by this author Kenjiro Kohri More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Shear stress-mediated upregulation of GTP cyclohydrolase/tetrahydrobiopterin pathway ameliorates hypertension-related decline in reendothelialization capacity of endothelial progenitor cells.

Guanosine triphosphate cyclohydrolase/tetrahydrobiopterin (GTPCH)/(BH4) pathway has been proved to regulate the function of endothelial progenitor cells (EPCs) in deoxycorticosterone acetate-salt hypertensive mice, indicating that GTPCH/BH4 pathway may be an important repair target for hypertension-related endothelial injury. Shear stress is an important nonpharmacologic strategy to modulate the function of EPCs. Here, we investigated the effects of laminar shear stress on the GTPCH/BH4 pathway and endothelial repair capacity of circulating EPCs in hypertension. Laminar shear stress was loaded on the human EPCs from hypertensive patients and normotensive patients. The in-vitro function, in-vivo reendothelialization capacity and GTPCH/BH4 pathway of human EPCs were evaluated. Both in-vitro function and reendothelialization capacity of EPCs were lower in hypertensive patients than that in normotensive patients. The GTPCH/BH4 pathway of EPCs was downregulated in hypertensive patients. Shear stress increased in-vitro function and reendothelialization capacity of EPCs from hypertensive patients and normotensive patients. Furthermore, shear stress upregulated the expression of GTPCH I and levels of BH4, nitric oxide, and cGMP of EPCs, and reduced thrombospondin-1 expression. With treatment of GTPCH knockdown or nitroarginine methyl ester inhibition, shear stress-induced increased levels of BH4, nitric oxide and cGMP of EPCs was suppressed. When GTPCH/BH4 pathway of EPCs was blocked, the effects of shear stress on in-vitro function and reendothelialization capacity of EPCs were inhibited. The study demonstrates for the first time that shear stress-induced upregulation of the GTPCH/BH4 pathway ameliorates hypertension-related decline in endothelial repair capacity of EPCs. These findings provide novel nonpharmacologic therapeutic approach for hypertension-related endothelial repair.

Oral chromium picolinate impedes hyperglycemia-induced atherosclerosis and inhibits proatherogenic protein TSP-1 expression in STZ-induced type 1 diabetic ApoE\u2212/\u2212 mice

Increasing evidence suggests thrombospondin-1 (TSP-1), a potent proatherogenic matricellular protein, as a putative link between hyperglycemia and atherosclerotic complications in diabetes. We previously reported that the micronutrient chromium picolinate (CrP), with long-standing cardiovascular benefits, inhibits TSP-1 expression in glucose-stimulated human aortic smooth muscle cells in vitro. Here, we investigated the atheroprotective action of orally administered CrP in type 1 diabetic apolipoprotein E-deficient (ApoE−/−) mice and elucidated the role of TSP-1 in this process. CrP decreased lipid burden and neointimal thickness in aortic root lesions of hyperglycemic ApoE−/− mice; also, smooth muscle cell (SMC), macrophage and leukocyte abundance was prevented coupled with reduced cell proliferation. Attenuated lesion progression was accompanied with inhibition of hyperglycemia-induced TSP-1 expression and reduced protein O-glycosylation following CrP treatment; also, PCNA and vimentin (SMC synthetic marker) expression were reduced while SM-MHC (SMC contractile marker) levels were increased. To confirm a direct role of TSP-1 in diabetic atherosclerosis, hyperglycemic TSP-1−/−/ApoE−/− double knockout mice were compared with age-matched hyperglycemic ApoE−/− littermates. Lack of TSP-1 prevented lesion formation in hyperglycemic ApoE−/− mice, mimicking the atheroprotective phenotype of CrP-treated mice. These results suggest that therapeutic TSP-1 inhibition may have important atheroprotective potential in diabetic vascular disease.

FGF7/FGFR2 signal promotes invasion and migration in human gastric cancer through upregulation of thrombospondin-1.

Fibroblast growth factor 7 (FGF7) is a mesenchyme-specific heparin-binding growth factor that binds FGF receptor 2 (FGFR2) to regulate numerous cellular and physiological processes. FGF7/FGFR2 signal is associated with gastric cancer progression. In the present study, we investigated the molecular mechanism by which FGF7/FGFR2 promotes invasion and migration in human gastric cancer. We first demonstrated that increased FGFR2 expression in human gastric cancer tissues was significantly associated with tumor depth and clinical stage in human gastric cancer tissues. Thrombospondin 1 (THBS1) is an extracellular glycoprotein that plays multiple roles in cell-matrix and cell-cell interactions. Increased expression of THBS1 significantly correlated with tumor differentiation. FGFR2 and THBS1 expression were both increased in cancer tissues as compared with adjacent normal tissues and their expression was positively correlated. In vitro, FGF7 stimulation of cell invasion and migration was partially suppressed by the FGFR2 knockdown. In addition, FGF7/FGFR2 upregulated THBS1, and cell invasion and migration were decreased by knockdown of THBS1. Furthermore, the PI3K/Akt/mTOR signaling pathway was predominantly responsible for FGF7/FGFR2-induced THBS1 upregulation. Taken together, our data suggest that FGF7/FGFR2/THBS1 is associated with the regulation of invasion and migration in human gastric cancer.

Perindopril Induces TSP-1 Expression in Hypertensive Patients with Endothelial Dysfunction in Chronic Treatment.

Thrombospondin-1 (TSP-1) is a potent endogenous inhibitor of both physiological and pathological angiogenesis, widely studied as a target in drug development for treating cancer. Several studies performed in the cardiovascular field on TSP-1 are contradictory, the role of TSP-1 in the physiopathology of cardiovascular disorders (CVDs) being, for the moment, incompletely understood and may be due to the presence of several domains in its structure which can stimulate many cellular receptors. It has been reported to inhibit NO-mediated signaling and to act on the angiogenesis, tissue perfusion, endothelial cell proliferation, and homeostasis, so we aimed to quantify the effect Perindopril has on TSP-1 plasma levels in hypertensive patients with endothelial dysfunction in comparison with other antihypertensive drugs, such as beta blockers, calcium channel blockers, and diuretics, in a chronic treatment. As a conclusion, patients under treatment with Perindopril had increased plasma levels of TSP-1 compared with other hypertensive patients and with the control group. The results of this study confirms the pleiotropic properties of Perindopril: anti-proliferative, anti-inflammatory, with effects showed by quantifying a single biomarker: TSP-1.