Thrombospondin-1 Protein Expression Research Articles

O-GlcNAcylation regulates angiotensin II-dependent induction of thrombospondin 1

O-Linked attachment of β-N-acetylglucosamine (O-GlcNAc) on serine and threonine residues of nuclear, cytoplasmic, and mitochondrial proteins is a highly dynamic and ubiquitous post-translational modification that plays an important role in altering the function, activity, subcellular localization, and stability of target proteins. It has been proposed that physiologically, O-GlcNAcylation serves primarily to modulate cellular signaling and transcription regulatory pathways in response to nutrients and stress. However, in the vascular physiology literature, O-GlcNAc has predominantly been shown to be detrimental. Our preliminary data has shown that O-GlcNAcylation can cause aortic stiffness, however, the mechanisms underlying this phenotype remain to be elucidated. The predominant mechanisms by which O-GlcNAcylation contributes to alterations in proteostasis include competition with other post-translational modifications as well as regulation of gene expression. Thrombospondin-1 (TSP1) is a multifunctional matrix glycoprotein that impacts extracellular function (ECM) and cell adhesion, migration, proliferation, and cell-cell interaction. Therefore, we hypothesized that TSP1 could be an important downstream mediator of O-GlcNAcylation in aortic vascular smooth muscle cells (VSMCs). To test this hypothesis, primary VSMCs from the thoracic aorta were isolated from 12-week-old female Wistar rats and submitted to angiotensin (ANG) II treatment (100 nM, 3h). Some cells were also treated in the presence of ANG II type 1 (AT1)-receptor antagonist losartan (10−5 M, 30 min). ANG II was chosen because it is a physiological stressor known to cause arterial stiffening. O-GlcNAcylation (RL2 antibody), O-GlcNAcase (OGA), and TSP1 protein amounts were analyzed by Western blot, and the results were expressed in arbitrary units (AU) and mean ±standard error mean. Unpaired Student's test T was used and p<0.05 was considered statistically significant. Genetic and pharmacological inhibition of O-GlcNAc was achieved through OGT silencing and inhibition with a shRNA (FunGene6) and OSMI-4 (10−5 M, 30 min), respectively. As hypothesized, ANG II significantly increased O-GlcNAc in aortic VSMC (0.3±0.03 vs 0.5±0.03 AU n=8, p<0.05) which was inhibited by losartan (0.1±0.02). Moreover, ANG II significantly decreased OGA expression (0.16±0.04 vs 1.16±0.19 AU n=4, p<0.05), the enzyme responsible for removing O-GlcNAc motifs, supporting the observed increase in O-GlcNAc expression. Importantly, ANG II also increased TSP1 protein expression (0.2±0.04 vs 2.0±0.4 n=4, p<0.05), however, shRNA (0.7±0.05) for OGT and OSMI-4 (0.06±0.08) prevented this increase. Overall, these data indicate that Ang II-AT1R signaling can cause O-GlcNAcylation and that TSP1 could be an important downstream mediator of O-GlcNAc-mediated aortic stiffness. National Institutes of Health (R00HL151889, P20GM109091-Pilot Project, and P20GM103641-Pilot Project). University of South Carolina Offce of Research ASPIRE Award (180950-23-64136). American Heart Association Postdoctoral Fellow (916031). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Skin-derived precursor conditioned medium alleviated photoaging via early activation of TGF-β/Smad signaling pathway by thrombospondin1: In vitro and in vivo studies

Photoaging is one major exogenous factor of skin aging. Efficacy and safety of current anti-photoaging therapies remained to be improved. Our previous studies indicated that skin-derived precursors (SKPs) alleviated photodamage by early activation of TGF-β/Smad signaling pathway via thrombospondin1 (TSP1). However, the research concerning SKP conditioned medium (SKP-CM) has never been reported. In the current study, we aimed to explore the anti-photoaging effects of SKP-CM both in vitro and in vivo, and to elucidate the possible mechanisms. Mouse SKP-CM (mSKP-CM) collection was optimized by a comparative method. The concentration of protein and growth factors in mSKP-CM was detected using BCA protein assay kit and growth factor protein chip. The anti-photoaging effects of mSKP-CM and its regulation of key factors in the TGF-β/Smad signaling pathway were explored using UVA + UVB photoaged mouse fibroblasts (mFBs) and nude mice dorsal skin. The research revealed that mSKP-CM contained significantly higher-concentration of protein and growth factors than mouse mesenchymal stem cell conditioned medium (mDMSC-CM). mSKP-CM alleviated mFBs photoaging by restoring cell viability and relieving senescence and death. ELISA, qRT-PCR, and western blot results implied the potential mechanisms were associated with the early activation of TGF-β/Smad signaling pathway by TSP1. In vivo experiments demonstrated that compared with the topical intradermal mDMSC-CM injection and retinoic acid cream application, the photodamaged mice dorsal skin intradermally injected with mSKP-CM showed significantly better improvement. Consistent with the in vitro results, both western blot and immunohistochemistry results confirmed that protein expression of TSP1, smad2/3, p-smad2/3, TGF-β1, and collagen I increased, and matrix metalloproteinases decreased. In summary, both in vitro and in vivo experiments demonstrated that mSKP-CM alleviated photoaging through an early activation of TGF-β/Smad signaling pathway via TSP1. SKP-CM may serve as a novel and promising cell-free therapeutical approach for anti-photoaging treatment and regenerative medicine.

4R-cembranoid suppresses glial cells inflammatory phenotypes and prevents hippocampal neuronal loss in LPS-treated mice.

Chronic neuroinflammation has been implicated in neurodegenerative disease pathogenesis. A key feature of neuroinflammation is neuronal loss and glial activation, including microglia and astrocytes. 4R-cembranoid (4R) is a natural compound that inhibits hippocampal pro-inflammatory cytokines and increases memory function in mice. We used the lipopolysaccharide (LPS) injection model to study the effect of 4R on neuronal density and microglia and astrocyte activation. C57BL/6J wild-type mice were injected with LPS (5 mg/kg) and 2 h later received either 4R (6 mg/kg) or vehicle. Mice were sacrificed after 72 h for analysis of brain pathology. Confocal images of brain sections immunostained for microglial, astrocyte, and neuronal markers were used to quantify cellular hippocampal phenotypes and neurons. Hippocampal lysates were used to measure the expression levels of neuronal nuclear protein (NeuN), inducible nitrous oxide synthase (iNOS), arginase-1, thrombospondin-1 (THBS1), glial cell-derived neurotrophic factor (GDNF), and orosomucoid-2 (ORM2) by western blot. iNOS and arginase-1 are widely used protein markers of pro- and anti-inflammatory microglia, respectively. GDNF promotes neuronal survival, and ORM2 and THBS1 are astrocytic proteins that regulate synaptic plasticity and inhibit microglial activation. 4R administration significantly reduced neuronal loss and the number of pro-inflammatory microglia 72 h after LPS injection. It also decreased the expression of the pro-inflammatory protein iNOS while increasing arginase-1 expression, supporting its anti-inflammatory role. The protein expression of THBS1, GDNF, and ORM2 was increased by 4R. Our data show that 4R preserves the integrity of hippocampal neurons against LPS-induced neuroinflammation in mice.

Scleral remodeling during myopia development in mice eyes: a potential role of thrombospondin-1

BackgroundScleral extracellular matrix (ECM) remodeling plays a crucial role in the development of myopia, particularly in ocular axial elongation. Thrombospondin-1 (THBS1), also known as TSP-1, is a significant cellular protein involved in matrix remodeling in various tissues. However, the specific role of THBS1 in myopia development remains unclear.MethodWe employed the HumanNet database to predict genes related to myopic sclera remodeling, followed by screening and visualization of the predicted genes using bioinformatics tools. To investigate the potential target gene Thbs1, we utilized lens-induced myopia models in male C57BL/6J mice and performed Western blot analysis to detect the expression level of scleral THBS1 during myopia development. Additionally, we evaluated the effects of scleral THBS1 knockdown on myopia development through AAV sub-Tenon’s injection. The refractive status and axial length were measured using a refractometer and SD-OCT system.ResultsDuring lens-induced myopia, THBS1 protein expression in the sclera was downregulated, particularly in the early stages of myopia induction. Moreover, the mice in the THBS1 knockdown group exhibited alterations in myopia development in both refraction and axial length changed compared to the control group. Western blotting analysis confirmed the effectiveness of AAV-mediated knockdown, demonstrating a decrease in COLA1 expression and an increase in MMP9 levels in the sclera.ConclusionOur findings indicate that sclera THBS1 levels decreased during myopia development and subsequent THBS1 knockdown showed a decrease in scleral COLA1 expression. Taken together, these results suggest that THBS1 plays a role in maintaining the homeostasis of scleral extracellular matrix, and the reduction of THBS1 may promote the remodeling process and then affect ocular axial elongation during myopia progression.

Thrombospondin-1 and prolyl 4-hydroxylase subunit alpha 3 as potential biomarkers of salivary gland fibrosis

Background/purposeFibrosis is present in various physiologic and pathologic conditions of salivary glands (SGs). This study aimed to identify novel biomarkers of SG fibrosis by next-generation sequencing. Materials and methodsWe established the SG fibrosis mouse model by excretory main duct ligation. Next-generation sequencing, differentially expressed gene (DEG) analysis, and gene set enrichment analysis was performed by comparing ligated and control SGs. We used algorithms of Cytohubba, molecular complex detection, Lasso logistic regression, and support vector machine to identify the key biomarkers. Selected key biomarkers were verified by the polymerase chain reaction and immunohistochemistry. We also retrieved and analyzed the key gene expression in the fibrosis of the heart, liver, lung, and kidney to ensure the generalization of key biomarkers in SG fibrosis. ResultsBoth interlobular and intralobular fibrosis was confirmed in the ligated SGs, with improved expressions of collagen I and transforming growth factor β. Next-generation sequencing identified 2666 upregulated DEGs and 336 downregulated DEGs, which were highly enriched in the extracellular matrix-related pathways. Multiple algorithms identified 15 key biomarkers in SG fibrosis, including Thrombospondin-1 (THBS1) and Prolyl 4-Hydroxylase Subunit Alpha 3 (P4HA3). The mRNA and protein expression of THBS1 and P4HA3 was verified in mice. THBS1 was also highly expressed in lung and kidney fibrosis, whereas P4HA3 was upregulated in liver fibrosis. ConclusionTHBS1 and P4HA3 may be potential biomarkers for SG fibrosis. They may be also applicable in the diagnosis of multi-organ fibrosis.

Thrombospondin-2 holds prognostic value and is associated with metastasis and the mismatch repair process in gastric cancer

BackgroundThis study aims to investigate thrombospondin 2 (TSP2) expression levels in gastric cancer (GC) and determine the relationship between TSP2 and clinical characteristics and prognosis.MethodsThe online database Gene Expression Profile Interactive Analysis (GEPIA) was used to analyse TSP2 mRNA expression levels in GC. The Kaplan–Meier plotter prognostic analysis tool was used to evaluate the influence of TSP2 expression on clinical prognosis in GC patients. TSP2 expression levels were analysed in paraffin-embedded GC samples and adjacent normal tissues by immunohistochemistry. The relationship between the clinicopathological characteristics and prognosis of GC patients was assessed. Transwell experiments were used to evaluate the effect of TSP2 on HGC27 and AGS cell invasion and migration. The EdU experiment was used to detect the effect of transfection of TSP2 on cell proliferation, and the flow cytometry experiment was used to detect the effect of TSP2 on cell apoptosis and the cell growth cycle. Western blotting (Wb) technology was used to detect MMP, E-cadherin, N-cadherin, Vimentin, Snail, AKT, PI3K, and VEGF protein expression in HGC27 cells.ResultsCompared with normal tissues, TSP2 mRNA expression in GC was significantly upregulated and was closely related to the clinical stage of GC. High TSP2 expression significantly affected the OS, FP and PPS of patients with GC. Among these patients, TSP2 expression levels did not affect the prognosis of patients with GC in the N0 subgroup but significantly affected the prognosis of patients with GC in the N (1 + 2 + 3) subgroup. TSP2 protein expression levels were significantly higher in GC tissue compared with normal tissues (P < 0.01). The overall survival (OS) and relapse-free survival (RFS) of patients with high TSP2 expression were lower than those of patients with low TSP2 expression. Cells transfected with the TSP2-silencing sequence exhibited increased apoptosis and inhibition of proliferation, migration and invasion. AKT and PI3K expression in cells was significantly downregulated (P < 0.01). AKT, PI3K and VEGF expression in cells transfected with the TSP2 silencing sequence was significantly reduced. Proliferation, migration, invasion ability, and TSP2 expression levels significantly correlated with mismatch repair genes, such as PMS2, MSH6, MSH2, and MLH1 (P < 0.05).ConclusionTSP2 expression is significantly increased in GC. TSP2 expression is closely related to metastasis and the mismatch repair process in GC patients and affects GC patient prognosis. The mechanism may involve regulating gastric cancer cell proliferation and migration by modulating the VEGF/PI3K/AKT signalling pathway. TSP2 is a potential marker and therapeutic target for the prognosis of GC patients.

Time course of capillary regression and an expression balance between vascular endothelial growth factor-A and thrombospondin-1 in the soleus muscle of hindlimb unloaded rats.

Skeletal muscle capillaries regress with disuse; however, information on time-dependent changes in the expression of pro- and anti-angiogenic factors in disused muscle is limited. This study aimed to clarify time-dependent changes in skeletal muscle capillarization, pro-angiogenic vascular endothelial growth factor-A (VEGF-A), and anti-angiogenic thrombospondin-1 (TSP-1) in the soleus muscle of hindlimb unloaded rat. Eight-week-old male Sprague Dawley rats were randomly divided into four groups corresponding to different hindlimb unloading (HU) duration at 0, 1, 2, and 3wk. Muscle atrophy and capillary regression worsened in the soleus muscle with longer periods of HU. The VEGF-A protein expression level was lower at week 1 than at week 0. In addition, the value at week 3 was also lower than those at weeks 0, 1, and 2. The TSP-1 protein expression level was higher at week 1 than that at week 0 but was similar at weeks 2 and 3. Moreover, reactive oxygen species, assessed by dihydroethidium fluorescence intensity on cryosection, were higher at weeks 2 and 3 than that at week 0. Depending on the HU period, VEGF-A and TSP-1 showed different expression patterns. In the early HU phase, TSP-1 may play an important role in capillary regression. However, when HU extends for a longer period, decreased VEGF-A, and/or increased oxidative stress may be more involved in capillary regression.

A2A receptor agonists and P2Y12 receptor antagonists modulate expression of thrombospondin-1 (TSP-1) and its secretion from Human Microvascular Endothelial Cells (HMEC-1).

To address the problem of resistance to standard antiplatelet therapy in some patients, our team proposed a purinoceptor-dependent dual therapy. Its efficacy is also determined by the condition of the vascular endothelium which, by secreting numerous factors, is involved in hemostasis. Among them, thrombospondin-1 is important in the context of thrombotic events. Therefore we sought to determine if the novel dual purinoceptor-dependent concept is associated with TSP-1 changes in vascular endothelial cells. TSP-1 expression in human microvascular endothelial cells was determined at transcriptional and protein level. We performed real-time PCR, the Western blot analysis and ELISA test. We found that TSP-1 mRNA and protein expression levels significantly changed in response to P1R agonists treatment. Furthermore, we have observed that co-administration of selective A2AR agonists (UK-432,097 or MRE0094) with P2Y12R antagonists altered TSP-1 expression levels, and the direction of these changes was not synergistic. MRE0094 applied with ARC69931MX or R-138727 increased mRNA expression from 39 to 56 or 57%, respectively (*P<0.05 vs. MRE0094; ***P<0.001 vs. control). Also, in the case of the P2Y12R antagonists used together with UK-432,097, there was an increase from 53 to 71 and 70% (*P<0.05 vs. UK-432,097; ***P<0.001 vs. control). The observed trends in gene expression were reflected in the protein expression and the level of its secretion from HMEC-1. The article presents evidence which proves that the purinoceptor-dependent concept is associated with TSP-1 changes in endothelial cells (EC). Moreover, Human Microvascular Endothelial Cells treatment applied together with agonists (MRE0094 or UK-432,097) and P2Y12R antagonist did not result in any synergistic effect, implicating a possible crosstalk between G proteins in GPCRs dependent signaling. Therefore, we suggest that understanding of the specific mechanism underlying TSP-1 alterations in EC in the context of the dual purinoceptor-dependent approach is essential for antiplatelet therapies and should be the subject of future research.

Peptidome characterization of ovarian cancer serum and the identification of tumor suppressive peptide ZYX36-58.

BackgroundSeveral serum biomarkers, including miRNA, mRNA, protein and peptides in cancer patients are also important mediators of cancer progression.MethodsThe differentially expressed peptides between the serum of ovarian cancer patients and healthy controls were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The function of the peptides was analyzed by CCK8, transwell, wound healing, and flow cytometry analysis. And the mechanism of the peptides was analyzed by peptide pull down, and high-throughput RNA-sequencing.ResultsA total of 7 and 46 peptides were significantly up-regulated and down-regulated in the serum of ovarian cancer patients, respectively. The precursor proteins of the differentially expressed peptides mainly involved in the complement and coagulation cascades, platelet activation, phagosome and focal adhesion pathways. Interestingly, focal adhesion, platelet activation, platelet-cancer cell interaction, complement activation, coagulation cascades and phagosome formation are all critical factors for cancer initiation or progression, which indicated that the peptides may play a crucial role in cancer development. And we identified one peptide, ZYX36-58, which was down-regulated in the serum of ovarian cancer patients, significantly inhibited invasion and migration and promoted the apoptosis of ovarian cancer cells. Mechanistic study indicated that ZYX36-58 interacted with and increased the protein level of the antiangiogenic protein thrombospondin-1 (TSP1), which has a tumor suppressive effect on ovarian cancer.ConclusionsZYX36-58, which was significantly down-regulated in the serum of ovarian cancer patients can significantly inhibit cell invasion, migration and promote apoptosis of ovarian cancer cells by binding and up-regulating TSP1 protein expression.

Mouse skin-derived precursors alleviates ultraviolet B irradiation damage via early activation of TGF-β/Smad pathway by thrombospondin1

ABSTRACT Our previous research implied mouse skin-derived precursors (mSKPs) possessed the capacity of anti-ultraviolet B (UVB) irradiation damage, and the mechanisms might be associated with transforming growth factor-β (TGF-β) signaling pathway activation. In this study, we investigated and compared the response to UVB irradiation between mSKPs and dermal mesenchymal stem cells (dMSCs), and explored the underlying mechanisms. Irradiation damage such as decreased cell viability, cell senescence, and cell death was observed in both mSKPs and dMSCs at 24 h after UVB exposure. In mSKPs, change in cell morphology, viability, cell senescence and death at the following time points implied the recovery of UVB irradiation damage. Additionally, thrombospondin1 (TSP1) and TGF-β1 increased significantly in mSKPs’ supernatant after UVB irradiation. The gene expression of TSP1, TGF-β1, metalloproteinase 1 (MMP1), and Collagen I elevated shortly after the UVB exposure. The protein expression of TSP1, TGF-β1, MMP1, Collagen I, smad2/3, and p-smad2/3 at multiple time points after the UVB exposure was consistent with the gene expression results. In dMSCs, no obvious recovery was noticed. Together, these results revealed that in mSKPs, one of the mechanisms to attenuate the UVB irradiation damage might be the early activation of TGF-β/Smad pathway by TSP1. Given that mSKPs could differentiate into fibroblast-like SKP-derived fibroblasts (SFBs) in vivo or with the presence of serum, mSKPs might serve as a therapeutic potential for fibroblasts supplement and UVB irradiation damage treatment. Abbreviations: SKPs: skin-derived precursors; mSKPs: mouse SKPs; UVB: ultraviolet B; TGF-β/Smad: transforming growth factor-β/Smad; TSP1: thrombospondin 1; MMP 13: metalloproteinases 13; TβRII: TGF-β receptor II; SFBs: SKP-derived fibroblasts; KEGG: Kyoto encyclopedia of genes and genomes; DEGs: differentially expressed genes; dMSCs: dermal mesenchymal stem cells; LM: light microscope; CCK-8: cell counting kit 8; ELISA: Enzyme-linked immuno sorbent assay; qRT-PCR: quantitative real-time polymerase chain reaction; TSPs: thrombospondins; ECM: extracellular matrix; R-smads: receptor-regulated smads

Thrombospondin-1 Exacerbates Acute Liver Failure and Hepatic Encephalopathy Pathology in Mice by Activating Transforming Growth Factor β1

Severe hepatic insults can lead to acute liver failure and hepatic encephalopathy (HE). Transforming growth factor β1 (TGFβ1) has been shown to contribute to HE during acute liver failure; however, TGFβ1 must be activated to bind its receptor and generate downstream effects. One protein that can activate TGFβ1 is thrombospondin-1 (TSP-1). Therefore, the aim of this study wastoassess TSP-1 during acute liver failure and HE pathogenesis. C57Bl/6 or TSP-1 knockout (TSP-1-/-) mice were injected with azoxymethane (AOM) to induce acute liver failure and HE. Liver damage, neurologic decline, and molecular analyses of TSP-1 and TGFβ1 signaling were performed. AOM-treated mice had increased TSP-1 and TGFβ1 mRNA and protein expression in the liver. TSP-1-/- mice administered AOM had reduced liver injury as assessed by histology and serum transaminase levels compared with C57Bl/6 AOM-treated mice. TSP-1-/- mice treated with AOM had reduced TGFβ1 signaling that was associated with less hepatic cell death as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and cleaved caspase 3 expression. TSP-1-/- AOM-treated mice had a reduced rate of neurologic decline, less cerebral edema, and a decrease in microglia activation in comparison with C57Bl/6 mice treated with AOM. Taken together, TSP-1 is an activator of TGFβ1 signaling during AOM-induced acute liver failure and contributes to both liver pathology and HE progression.

912 Mouse skin-derived precursors’ anti-UVB radiation damage through TGF-β/Smad pathway activation via thrombospondin 1

Former research indicated mouse skin-derived precursors (mSKPs) might have the capacity of anti-UVB radiation. The mechanisms might be associated with TGF-β/Smad pathway and thrombospondin 1 (TSP1). This study was to investigate mSKPs’ response to UVB radiation, and to study the role of TSP1 in TGF-β/Smad pathway in mSKPs’ anti-UVB radiation damage, as compared with dermal mesenchymal stem cells (DMSCs). Cell survival rate, senescence and death were determined. TSP1 and TGF-β1 in supernatant were tested. Gene and protein expression of TSP1, TGF-β1, MMP1, collagen I, smad2/3, and p-smad2/3 was measured. mSKPs’ survival rate decreased 24h after UVB radiation, but increased at 48h and 72h. DMSCs’ survival rate kept decreasing after radiation. Cell senescence and death 24h after radiation, but recovery at 48h were noticed in mSKPs. The recovery was not obvious in DMSCs. TSP1 and TGF-β1 in mSKPs’ supernatant increased 6h, 12h, and 24h after radiation (p<0.05). No significant change was noticed in DMSCs’ supernatant. In mSKPs, gene expression of TSP1, TGF-β1, MMP1, and collagen I increased 3h, 6h after radiation (p<0.01), decreased at 12h, 24h (p<0.01). In DMSCs, expression of TSP1 and collagen I decreased after radiation (p<0.05), expression of MMP1 increased after radiation (p<0.01), expression of TGF-β1 decreased 3h after radiation (p<0.01), increased at other time points (p<0.01). In mSKPs, protein expression of TSP1, TGF-β1, MMP1, and p-smad2/3 increased 6h, 12h, and 24h after radiation (p<0.05), expression of smad2/3 decreased after radiation (p<0.05). Expression of collagen I increased 6h, 12h after radiation (p<0.01), decreased at 24h (p<0.05). In DMSCs, expression of TGF-β1 and MMP1 increased after radiation (p<0.01), and expression of other proteins decreased (p<0.01). Radiation damage after UVB radiation were observed in mSKPs and DMSCs. The recovery was noticed in mSKPs, and one of the mechanisms might be the activation of TGF-β/Smad pathway by TSP1. In DMSCs, the recovery was not noticed, and there might be other mechanisms.

The immunoregulatory role of corneal epithelium-derived thrombospondin-1 in dry eye disease

PurposeIn this study, we examine the expression of corneal epithelium-derived thrombospondin-1 (TSP-1) and its immunomodulatory functions in a validated murine model of dry eye disease (DED). MethodsDED was induced in female C57BL/6 using a controlled environment chamber (CEC) for 14 days. mRNA and protein expression of TSP-1 by corneal epithelial cells was quantified using real-time PCR and flow cytometry. Corneal epithelial cells from either naïve or DED mice were cultured with bone marrow derived dendritic cells (BMDCs) in the presence of IFNγ for 48 h, and BMDC expression of MHC-II and CD86 was determined using flow cytometry. Next, either recombinant TSP-1 or anti-TSP-1 antibody was added to the co-culture, and BMDC expression of above activation markers was evaluated. Finally, either DED mice were topically treated with either recombinant TSP-1 or human serum albumin (HSA), and maturation of corneal DCs, expression of inflammatory cytokines, and DED severity were investigated. ResultsmRNA expression of TSP-1 by the corneal epithelium was upregulated in DED. Corneal epithelial cells derived from mice with DED demonstrated an enhanced capacity in suppressing BMDC expression of MHC-II and CD86 relative to wild type mice, and this effect was abrogated by TSP-1 blockade and potentiated by recombinant TSP-1. Finally, topical application of recombinant TSP-1 significantly suppressed corneal DC maturation and mRNA expression of pro-inflammatory cytokines, and ameliorated disease severity in mice with DED. ConclusionsOur study elucidates the function of epithelium-derived TSP-1 in inhibiting DC maturation and shows its translational potential to limit corneal epitheliopathy in DED.

Lentivirus media miR-1246 knockdown inhibits tumor growth and promotes apoptosis of SiHa cells

Objective: To study the effect of lentivirus-mediated microRNA (miR) -1246 RNA interference (RNAi) on biological characteristics and behaviors in cervical cancer cells as well as to identify the downstream signaling pathways affected. Methods: MiR-1246 specific cDNA was synthesized and cloned into the recombinant lentiviral vector (LV-miR-1246-inhibitor) . The SiHa cells were devided into three groups: no viral infection (negative control, NC) , infection with control virus (LV-NC) , and infection with miR-1246-inhibitor virus (LV-miR-1246-inhibitor) . The expression of the miR-1246 was detected by reverse transcription (RT) -PCR. Cell growth was analyzed by cell counting kit 8 (CCK-8) assay. The invasion was dectected by transwell matrige gel. Cell apoptosis was detected by flow cytometer. The growth of xenograft tumors was also investigated. Expression of thrombospondin-2 (THBS2) , matrix metalloproteinase (MMP) 2, 9 were also evaluated in the cells. Results: (1) The expression level of miR-1246 in SiHa cells (0.11±0.13) was significantly lower in group LV-miR-1246-inhibitor than those in the group LV-NC and the group NC (1.14±0.86 and 1.30±0.73, respectively; P<0.01) . (2) The proliferation of SiHa was also markedly suppressed in CCK-8 at 96 hours (P<0.01) . (3) The number of group LV-miR-1246-inhibitor was significantly less than those in the LV-NC and NC groups in transwell invasion assay (71±4, 162±5 and 188±5, respectively; P<0.01) . (4) The apoptosis rate of SiHa cells in the group LV-miR-1246-inhibitor [ (16.10±3.37) %] was significantly lower than those of group LV-NC and group NC [ (5.67±0.89) % and (1.78±0.08) %,P<0.01]. (5) The tumor volume in the nude mice group LV-miR-1246-inhibitor [ (287±59) mm(3)] was significantly lower than those in the LV-NC and NC groups [ (571±137) and (657±144) mm(3), respectively; P<0.01]. (6) Compared with the LV-NC group and the NC group, THBS2 protein expression in the tumor tissue of the nude mice in the group LV-miR-1246-inhibitor was significantly increased (P<0.05) , while the expression levels of MMP-2 and MMP-9 protein were significantly decreased (P<0.01) . Conclusion: These results suggest that miR-1246 functions during cervical cancer pathogenesis and tumor formation via the THBS2, MMP signaling pathway.

Abstract 230: Matrix Regulation of Lymphangiogenesis in Atherosclerosis

Background: Atherosclerosis is a consequence of an imbalance of cellular uptake of lipoproteins versus removal of cholesterol from the arterial wall. The lymphatic vasculature represents the primary route of cholesterol removal from atherosclerotic vessels. Previous studies have linked matrix protein thrombospondin-1 (TSP1), a well-known inhibitor of angiogenesis, with human atherosclerotic disease; however the mechanisms by which TSP1 contributes to atherosclerosis and its effect on arterial lymphangiogenesis remain unknown. Methods: The effect of TSP1 on human lymphatic endothelial cell (LEC) proliferation, migration and tube formation was investigated in vitro . TSP1 protein expression was analyzed in human and murine (ApoE -/- , 12 weeks Western diet) atherosclerotic aortic tissue and lesion free segments using Western blotting. The role of TSP1 and its cognate receptor CD47 in the pathogenesis of atherosclerosis was investigated using knockout animals. Results: TSP1 expression was significantly increased in both human and ApoE -/- atherosclerotic arteries compared to plaque free segments in human and wild type aortic tissue. TSP1 at physiologically relevant concentrations (11-22 nM, 24 hrs) inhibited LEC proliferation, migration, and tube formation. RT-PCR data demonstrated that CD47 is the predominant TSP1 receptor expressed in LEC (&gt; 50-fold over CD36 mRNA levels). Finally, genome-wide deficiency in TSP1 and CD47 receptor attenuated atherosclerotic lesion formation in the aorta and brachiocephalic artery by ~50% compared to wild type controls. Importantly, there was no difference in plasma cholesterol levels, body weight, fat and lean mass, fasting plasma glucose, and blood pressure between the experimental groups. Conclusions: The present study suggests that matrix protein TSP1 via CD47 receptor contributes to the pathogenesis of atherosclerosis. Inhibition of arterial lymphangiogenesis by TSP1-CD47 signaling may contribute to arterial lipid accumulation and atherosclerosis.

Effects of thrombospondin 1 down-regulation on biological function of lung adenocarcinoma A549 cells

Objective To observe the effect of down-regulation of platelet thrombospondin 1 (THBS1) on proliferation, apoptosis and cell cycle of lung adenocarcinoma A549 cells. Methods THBS1 protein expression in lung adenocarcinoma cell lines A549, H1299, H358, H1975, PC9, PG49 and Calu3 was detected by Western blotting. THBS1 small interfering RNA (siRNA) and control siRNA were transfected into A549 cells with the highest expression level of THBS1. The expression of THBS1 protein in THBS1 siRNA group, control group and transfected group was detected by Western blotting. Then cell counting kit-8 (CCK-8) kit was used to detect the proliferation of each group. Cell apoptosis and cell cycle distribution were analyzed by flow cytometry. Results The expression levels of THBS1 protein in different lung adenocarcinoma cell lines were different, of which A549 cells had the highest expression (1.120±0.087; F=358.087, P=0.000). Compared with non-transfected A549 cells and control group: The expression of THBS1 protein in A549 cells transfected with THBS1 siRNA (0.053±0.023) was significantly decreased (P=0.000), the cell proliferation ability was decreased(P=0.000), the apoptosis rates (THBS1 siRNA group: Early apoptosis rate, middle and late apoptosis rate and total apoptosis rate were (17.00±1.33)%, (3.70±0.56)% and (20.71±0.78)% were increased (F values were 115.899, 14.418 and 167.329 respectively, with P values of 0.000, 0.005 and 0.000 respectively), the ratio of G0/G1 phase cells (68.43±2.26)% was significantly increased (F=46.521, P=0.000), ratio of S phase cells (22.33±2.15)% was significantly increased(F=16.621, P=0.004) and G2/M phase cells (9.23±2.35)% decreased (F=9.282, P=0.015), the difference was statistically significant. Conclusion Silencing THBS1 gene and decreasing the expression of THBS1 protein in lung adenocarcinoma A549 cells can weaken cell proliferation ability, promote cell apoptosis and inhibit the cell cycle progression. Key words: Thrombospondin 1; Lung adenocarcinoma; Cell proliferation; Apoptosis; Cell cycle

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.

Properdistatin inhibits angiogenesis and improves vascular function in human melanoma xenografts with low thrombospondin-1 expression.

In this study, the effect of properdistatin, a novel peptide derived from the thrombospondin 1 (TSP-1) domain of properdin, was investigated in three melanoma xenograft models with different TSP-1 expression. The tumors were grown in dorsal window chambers and were treated with 80 mg/kg/day properdistatin or vehicle. Morphological parameters of the tumor vasculature were assessed from high resolution transillumination images. Blood supply time (i.e., the time required for arterial blood to flow from a supplying artery to downstream microvessels) and plasma velocities were assessed from first-pass imaging movies recorded after a bolus of fluorescence-labeled dextran had been administered intravenously. Gene and protein expression of TSP-1 were assessed with quantitative PCR and immunohistochemistry, respectively. Properdistatin treatment inhibited angiogenesis in low TSP-1 expressing tumors but did not alter the vasculature in high TSP-1 expressing tumors. In low TSP-1 expressing tumors, properdistatin selectively removed small-diameter capillaries, but did not change the morphology of tumor arterioles or tumor venules. Properdistatin also reduced blood supply times and increased plasma velocities, implying that the treatment reduced the geometric resistance to blood flow and improved vascular function.

Intraluminal delivery of thrombospondin-2 small interfering RNA inhibits the vascular response to injury in a rat carotid balloon angioplasty model.

In an effort to inhibit the response to vascular injury that leads to intimal hyperplasia, this study investigated the in vivo efficacy of intraluminal delivery of thrombospondin-2 (TSP-2) small interfering RNA (siRNA). Common carotid artery (CCA) balloon angioplasty injury was performed in rats. Immediately after denudation, CCA was transfected intraluminally (15 min) with one of the following: polyethylenimine (PEI)+TSP-2 siRNA, saline, PEI only, or PEI+control siRNA. CCA was analyzed at 24 h or 21 d by using quantitative real-time PCR and immunohistochemistry. TSP-2 gene and protein expression were significantly up-regulated after endothelial denudation at 24 h and 21 d compared with contralateral untreated, nondenuded CCA. Treatment with PEI+TSP-2 siRNA significantly suppressed TSP-2 gene expression (3.1-fold) at 24 h and TSP-2 protein expression, cell proliferation, and collagen deposition up to 21 d. These changes could be attributed to changes in TGF-β and matrix metalloproteinase-9, the downstream effectors of TSP-2. TSP-2 knockdown induced anti-inflammatory M2 macrophage polarization at 21 d; however, it did not significantly affect intima/media ratios. In summary, these data demonstrate effective siRNA transfection of the injured arterial wall and provide a clinically effective and translationally applicable therapeutic strategy that involves nonviral siRNA delivery to ameliorate the response to vascular injury.-Bodewes, T. C. F., Johnson, J. M., Auster, M., Huynh, C., Muralidharan, S., Contreras, M., LoGerfo, F. W., Pradhan-Nabzdyk, L. Intraluminal delivery of thrombospondin-2 small interfering RNA inhibits the vascular response to injury in a rat carotid balloon angioplasty model.

Astrocyte-secreted thrombospondin-1 modulates synapse and spine defects in the fragile X mouse model.

Astrocytes are key participants in various aspects of brain development and function, many of which are executed via secreted proteins. Defects in astrocyte signaling are implicated in neurodevelopmental disorders characterized by abnormal neural circuitry such as Fragile X syndrome (FXS). In animal models of FXS, the loss in expression of the Fragile X mental retardation 1 protein (FMRP) from astrocytes is associated with delayed dendrite maturation and improper synapse formation; however, the effect of astrocyte-derived factors on the development of neurons is not known. Thrombospondin-1 (TSP-1) is an important astrocyte-secreted protein that is involved in the regulation of spine development and synaptogenesis. In this study, we found that cultured astrocytes isolated from an Fmr1 knockout (Fmr1 KO) mouse model of FXS displayed a significant decrease in TSP-1 protein expression compared to the wildtype (WT) astrocytes. Correspondingly, Fmr1 KO hippocampal neurons exhibited morphological deficits in dendritic spines and alterations in excitatory synapse formation following long-term culture. All spine and synaptic abnormalities were prevented in the presence of either astrocyte-conditioned media or a feeder layer derived from FMRP-expressing astrocytes, or following the application of exogenous TSP-1. Importantly, this work demonstrates the integral role of astrocyte-secreted signals in the establishment of neuronal communication and identifies soluble TSP-1 as a potential therapeutic target for Fragile X syndrome.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0256-9) contains supplementary material, which is available to authorized users.