Relationship of Thrombospondin 1 to von Willebrand Factor and ADAMTS-13 in Sickle Cell Disease Patients of Arab Ethnicity.

BACKGROUND: Thrombospondin 1 (TSP-1) is a multifunctional glycoprotein secreted by platelets. In sickle cell disease (SCD), TSP-1 promotes red cell adhesion to the endothelium by binding to von Willebrand factor (vWF) and inhibiting its degradation by the protease ADAMTS-13. We investigated a possible correlation between TSP-1, vWF and ADAMTS-13 in adult and pediatric SCD patients. METHODS: Using commercially available ELISA kits, TSP-1, vWF and ADAMTS-13 levels were measured in 59 SCD patients (20 children and 39 adults) and compared with 59 age- and sex-matched controls. Associations between TSP-1 and parameters of interest were analyzed using Pearson's correlation coefficient. RESULTS: Although TSP-1 levels were higher in adult and pediatric SCD patients than in controls, the increase was not statistically significant (p > 0.05). We found a significant positive correlation between TSP-1 and platelet count in both adult (r = 0.402, p = 0.01) and pediatric (r = 0.589, p = 0.01) patients, which is expected due to increased platelet activation in SCD. There was a positive correlation between TSP-1 and vWF in normal adults (r = 0.305, p = 0.049) and children (r = 0.633, p = 0.005) but not in patients (p > 0.05). A significant negative correlation between TSP-1 and ADAMTS-13 activity (r = -0.41, p = 0.01) was found in adult patients. Also, a significant negative correlation between TSP-1 and ADAMTS-13/vWF antigen ratio in both normal controls (r = -0.595, p = 0.009) and patients (r = -0.493, p = 0.032) is reported for the pediatric group. CONCLUSIONS: Our findings confirm the inhibitory effects of TSP-1 on ADAMTS-13 activity in adult SCD patients. The negative correlation reported between TSP-1 and ADAMTS-13/vWF antigen ratio in pediatric subjects suggests a possible protective mechanism in younger individuals, although this is not related to the presence of SCD. This work emphasizes the impact of age on interpreting results related to the regulation of vWF expression and interaction with TSP-1 and ADAMTS-13 in SCD.

Protein Concentrations of Thrombospondin-1, MIP-1ß, and S100A8 Suggest the Reflection of a Pregnancy Clock in Mid-Trimester Amniotic Fluid.

The development of immunoassays enables more sophisticated studies of the associations between protein concentrations and pregnancy outcomes, allowing early biomarker identification that can improve neonatal outcomes. The aim of this study was to explore associations between selected mid-trimester amniotic fluid proteins and (1) overall gestational duration and (2) spontaneous preterm delivery. A prospective cohort study, including women undergoing mid-trimester transabdominal genetic amniocentesis, was performed in Gothenburg, Sweden, 2008-2016 (n = 1072). A panel of 27 proteins related to inflammation was analyzed using Meso-Scale multiplex technology. Concentrations were adjusted for gestational age at sampling, experimental factors, year of sampling, and covariates (maternal age at sampling, parity (nulliparous/multiparous), smoking at first prenatal visit, and in vitro fertilization). Cox regression analysis of the entire cohort was performed to explore possible associations between protein concentrations and gestational duration. This was followed by Cox regression analysis censored at 259 days or longer, to investigate whether associations were detectable in women with spontaneous preterm delivery (n = 47). Finally, linear regression models were performed to analyze associations between protein concentrations and gestational duration in women with spontaneous onset of labor at term (n = 784). HMG-1, IGFBP-1, IL-18, MIP-1α, MIP-1ß, S100A8, and thrombospondin-1 were significantly associated with gestational duration at term, but not preterm. Increased concentrations of thrombospondin-1, MIP-1ß, and S100A8, respectively, were significantly associated with decreased gestational duration after the Holm-Bonferroni correction in women with spontaneous onset of labor at term. This adds to the concept of a pregnancy clock, where our findings suggest that such a clock is also reflected in the amniotic fluid at early mid-trimester, but further research is needed to confirm this.

ADAM (a Disintegrin and Metalloproteinase) 15 Deficiency Exacerbates Ang II (Angiotensin II)-Induced Aortic Remodeling Leading to Abdominal Aortic Aneurysm.

OBJECTIVE: ADAM (a disintegrin and metalloproteinase) 15-a membrane-bound metalloprotease from the ADAM (disintegrin and metalloproteinase) family-has been linked to endothelial permeability, inflammation, and metastasis. However, its function in aortic aneurysm has not been explored. We aimed to determine the function of ADAM15 in the pathogenesis of aortic remodeling and aneurysm formation. Approach and Results: Male Adam15-deficient and WT (wild type) mice (10 weeks old), on standard laboratory diet, received Ang II (angiotensin II; 1.5 mg/kg per day) or saline (Alzet pump) for 2 or 4 weeks. Ang II increased ADAM15 in WT aorta, while Adam15-deficiency resulted in abdominal aortic aneurysm characterized by loss of medial smooth muscle cells (SMCs), elastin fragmentation, inflammation, but unaltered Ang II-mediated hypertension. In the abdominal aortic tissue and primary aortic SMCs culture, Adam15 deficiency decreased SMC proliferation, increased apoptosis, and reduced contractile properties along with F-actin depolymerization to G-actin. Ang II triggered a markedly greater increase in THBS (thrombospondin) 1 in Adam15-deficient aorta, primarily the medial layer in vivo, and in aortic SMC in vitro; increased SSH1 (slingshot homolog 1) phosphatase activity and cofilin dephosphorylation that promoted F-actin depolymerization and G-actin accumulation. rhTHBS1 (recombinant THBS1) alone was sufficient to activate the cofilin pathway, increase G-actin, and induce apoptosis of aortic SMCs, confirming the key role of THBS1 in this process. Further, in human abdominal aortic aneurysm specimens, decreased ADAM15 was associated with increased THBS1 levels and loss of medial SMCs. CONCLUSIONS: This study is the first to demonstrate a key role for ADAM15 in abdominal aortic aneurysm through regulating the SMC function, thereby placing ADAM15 in a critical position as a potential therapeutic target for abdominal aortic aneurysm.

Losartan treatment significantly attenuates the fibrotic changes in the corpus cavernosum of castrated rats.

INTRODUCTION AND OBJECTIVES: The aim of this study is to evaluate the effects of castration and subsequent losartan administration on the fibrosis-related parameters in the corpora cavernosa of castrated rats. MATERIAL AND METHODS: Twenty-four male rats were divided into four equal groups. Group 1:sham surgery plus vehicle (0.9% NaCl) (control:con), group 2:sham surgery plus losartan (con+los), group 3: castration plus vehicle (castration:cast) and group 4:castration plus losartan (cast+los). After four weeks of oral losartan treatment, corporal levels of transforming growth factor-beta (TGF-ß), thrombospondin-1 (TSP-1), alpha-actin, beta-actin and fibronectin were investigated by ELISA kits. Changes in the collagen and smooth muscle content were evaluated by histological analysis with Masson trichrome staining. RESULTS: Initial and post-treatment body weights of rats were similar among groups. Castration significantly increased the expression of TGF-ß, TSP-1 and fibronectin and resulted in a significant decrease in alpha-actin levels in the corpora cavernosa. Administration of losartan reduced the levels of TGF-ß, TSP-1 and fibronectin in castrated rats. Alpha actin levels also increased after losartan treatment. Beta-actin levels were not significantly different among 4 groups. The levels of all markers were similar in group 1 and 2. Rate of fibrosis was significantly higher in castrated rats and treatment with losartan reduced this rate. CONCLUSION: Castration increased the expression of fibrosis-related markers in the corpora cavernosa of rats. Administration of losartan significantly attenuated those changes and exerted an antifibrotic effect.

Thrombospondin 1 and Its Diverse Roles as a Regulator of Extracellular Matrix in Fibrotic Disease.

Thrombospondin 1 (TSP1) is a matricellular extracellular matrix protein that has diverse roles in regulating cellular processes important for the pathogenesis of fibrotic diseases. We will present evidence for the importance of TSP1 control of latent transforming growth factor beta activation in renal fibrosis with an emphasis on diabetic nephropathy. Other functions of TSP1 that affect renal fibrosis, including regulation of inflammation and capillary density, will be addressed. Emerging roles for TSP1 N-terminal domain regulation of collagen matrix assembly, direct effects of TSP1-collagen binding, and intracellular functions of TSP1 in mediating endoplasmic reticulum stress responses in extracellular matrix remodeling and fibrosis, which could potentially affect renal fibrogenesis, will also be discussed. Finally, we will address possible strategies for targeting TSP1 functions to treat fibrotic renal disease.

Trimetallic signal amplification aptasensor for TSP-1 detection based on Ce-MOF@Au and AuPtRu nanocomposites.

In this work, an aptamer was used as the target capturing agent and a trimetallic signal amplification strategy based on Ce-MOF@Au and AuPtRu NPs was demonstrated for the sensitive detection of TSP-1. Herein, the synthesized AuPtRu nanocomposite (AuPtRu NPs) not only acts as the catalyst for catalyzing hydrogen peroxide but also acts as a nanocarrier for capturing the -NH2 termination single strand DNA (S1) to obtain the signal probe (SP, AuPtRu nanocomposite/S1). Then, SP was efficiently linked into TSP-1 aptamers with the addition of complementary linking strands to form M1 (SP/aptamer). The Ce-MOF@Au nanocomposites were obtained by in situ reduction and used as GCE electrode modification materials. The -NH2-modified capture probe (CP) DNA was immobilized on the surface of Ce-MOF@Au nanocomposites for hybridizing SP. In the presence of the target TSP-1, the aptamer recognizes the target and binds strongly so that SP is released from the prepared M1 and then hybridized with CP. When the detection solution contains an electrochemical matrix of H2O2, AuPtRu NPs can oxidize H2O2 to obtain an enhanced signal. Furthermore, the proposed aptasensor has a very low LOD of 0.13 fg mL-1 TSP-1 in the detection range of 1 fg mL-1 to 10 ng mL-1. Moreover, the proposed platform also has application implications for other potential targets.

SILAC-based quantitative MS approach for real-time recording protein-mediated cell-cell interactions.

In tumor microenvironment, interactions among multiple cell types are critical for cancer progression. To understand the molecular mechanisms of these complex interplays, the secreted protein analysis between malignant cancer cells and the surrounding nonmalignant stroma is a good viewpoint to investigate cell-cell interactions. Here, we developed two stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry (MS)/MS approaches termed spike-in SILAC and triple-SILAC to quantify changes of protein secretion level in a cell co-cultured system. Within the co-culture system of CT26 and Ana-1 cells, the spike-in SILAC and triple-SILAC MS approaches are sensitive to quantitatively measure protein secretion changes. Three representative quantified proteins (Galectin-1, Cathepsin L1 and Thrombospondin-1) by two SILAC-based MS methods were further validated by Western blotting, and the coming result matched well with SILACs'. We further applied these two SILACs to human cell lines, NCM460 and HT29 co-culture system, for evaluating the feasibility, which confirmed the spike-in and triple SILAC were capable of monitoring the changed secreted proteins of human cell lines. Considering these two strategies in time consuming, sample complexity and proteome coverage, the triple-SILAC way shows more efficiency and economy for real-time recording secreted protein levels in tumor microenvironment.

Quantification of the Extracellular Matrix Molecule Thrombospondin 1 and Its Pericellular Association in the Brain Using a Semiautomated Computerized Approach.

The structure and functions of the extracellular matrix (ECM), its spatial distribution and pericellular association of ECM molecules remain poorly understood. Colocalization of ECM molecules with cell phenotypes through immunohistochemistry can provide crucial insights into their juxtacrine signaling role as well as their structural relevance to tissue architecture. As manual quantification of images introduces intra- and inter-user bias and is cumbersome for high-throughput approaches, we implemented an automated high-throughput method to quantify the spatial distribution and cellular association of one ECM molecule, thrombospondin 1 (TSP1) with two major cell phenotypes, neurons, and astrocytes. The distribution of TSP1 was homogeneous throughout the striatum and cortex along the anterior-posterior axis. TSP1 occupied 8.85% of the striatum and 7.40% in the cortex. TSP1 also associated with 94.58% and 88.45% of neurons in the striatum and cortex. The association with astrocytes was significantly lower at 47.55% and 28.09%. These findings highlight the key role that TSP1 plays in neuron physiology in a healthy brain, but also highlights key regional difference in astrocytes secreting ECM molecules. The semiautomated approach implemented here will improve the throughput and reliability of measuring the distribution and cellular colocalization of ECM molecules.

Transcriptional and Post-Transcriptional Regulation of Thrombospondin-1 Expression: A Computational Model.

Hypoxia is an important physiological stress signal that drives angiogenesis, the formation of new blood vessels. Besides an increase in the production of pro-angiogenic signals such as vascular endothelial growth factor (VEGF), hypoxia also stimulates the production of anti-angiogenic signals. Thrombospondin-1 (TSP-1) is one of the anti-angiogenic factors whose synthesis is driven by hypoxia. Cellular synthesis of TSP-1 is tightly regulated by different intermediate biomolecules including proteins that interact with hypoxia-inducible factors (HIFs), transcription factors that are activated by receptor and intracellular signaling, and microRNAs which are small non-coding RNA molecules that function in post-transcriptional modification of gene expression. Here we present a computational model that describes the mechanistic interactions between intracellular biomolecules and cooperation between signaling pathways that together make up the complex network of TSP-1 regulation both at the transcriptional and post-transcriptional level. Assisted by the model, we conduct in silico experiments to compare the efficacy of different therapeutic strategies designed to modulate TSP-1 synthesis in conditions that simulate tumor and peripheral arterial disease microenvironment. We conclude that TSP-1 production in endothelial cells depends on not only the availability of certain growth factors but also the fine-tuned signaling cascades that are initiated by hypoxia.

High expression of intratumoral stromal proteins is associated with chemotherapy resistance in breast cancer.

We studied the changes of intratumoral stromal proteins including THBS1, TNC, FN, SPARC and α-SMA, following neoadjuvant chemotherapy. The underlying mechanisms by which THBS1 and TNC regulated resistance to docetaxel were further studied using functional studies. 100 patients with newly diagnosed breast cancer were treated with alternating sequential doxorubicin and docetaxel. Immunohistochemistry (IHC) staining for stromal proteins was performed on pre- and post-treatment core biopsies respectively. THBS1 and TNC were further validated with IHC in an independent cohort of 31 patients. A high baseline combined expression score of the 5 stromal proteins predicted independently for poor progression-free (HRadjusted 2.22, 95% CI 1.06-4.64) and overall survival (HRadjusted 5.94, 95% CI 2.25-15.71). After 1-2 cycles of chemotherapy, increased expression of THBS1, TNC, FN, SPARC and α-SMA was seen in patients with subsequent pathological lymph node involvement at surgery. Increased expression of THBS1 and TNC compared to baseline was also seen in intrinsically resistant tumors, but not in sensitive ones. Both THBS1 and TNC-associated chemoresistance were confirmed in an independent validation cohort. Exogenous THBS1 and TNC protected MCF-7 cells against proliferation inhibition induced by docetaxel through activating integrin ß1/mTOR pathway. Thus, up-regulation of THBS1, TNC, FN, SPARC and α-SMA following neoadjuvant chemotherapy was associated with chemotherapy resistance in breast cancer patients. Functional studies showed THBS1 and TNC to mediate chemoresistance through the integrin ß1/mTOR pathway, suggesting that therapies targeting integrin ß1/mTOR pathway may be a promising strategy to overcome chemotherapy resistance.

Glioma-derived thrombospondin-1 modulates cd14+ cell tolerogenic properties.

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

Net platelet angiogenic activity (NPAA) correlates with progression and prognosis of non-small cell lung cancer.

Circulating platelets are abundant sources of angiogensis molecules for the tumor vasculature affecting tumor growth and metastasis. The relationship between non-small cell lung cancer (NSCLC) and intra-platelet levels of VEGF, TSP-1 and net platelet angiogenic activity (NPAA) is unclear. The aim of this study was to better understand the role of these factors in the progression of NSCLC cancer and to assess its clinical significance. Platelet VEGF and TSP-1 and NPAA were measured preoperatively in 68 patients with NSCLC by ELISA or Capillary tube formation assay. VEGF, TSP-1 and NPAA distributions in cancer patients and healthy volunteers were compared using the Mann-Whitney U test. The Kaplan-Meier method, univariate and multivariate regression analysis was used to analyze the correlation between these factors and clinicopathological features, overall survival and disease-free survival. Mean intra-platelet TSP-1 level was slightly higher in patients than in healthy subjects (p = 0.092). Intra-platelet TSP-1 levels were significantly higher in patients with involvement greater than T2 or stage III, compared to other patients. Mean intra-platelet VEGF level was 40.8 pg/106 in patients compared to 21.9 ng/106 in healthy subjects (p = 0.041). Median value of NPAA in patients was significantly higher than that in healthy controls (p<0.001). Patients with high NPAA are more likely to exhibit aggressive clinical pathological features. NPAA greater than the median are associated with poor prognosis. The elevated NPAA have better correlation with tumor microvessel density (MVD) than platelet-derived VEGF. The areas under receiver operating curve (AUROC) of NPAA were higher than that of platelet derived VEGF in different groups. A multivariate analysis showed that NPAA are independent prognostic factors. These results indicated that NPAA may be a clinically useful indicator for diagnostic and prognostic evaluation in NSCLC patients.

Thrombospondin-1 is highly expressed in desmoplastic components of invasive ductal carcinoma of the breast and associated with lymph node metastasis.

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

An autologous platelet-rich plasma stimulates periodontal ligament regeneration.

BACKGROUND: Regeneration of periodontal tissues is one of the most important goals for the treatment of periodontal disease. The technology of plasma rich in growth factors provides a biologic approach for the stimulation and acceleration of tissue healing. The purpose of this study is to evaluate the biologic effects of this technology on primary human periodontal ligament fibroblasts. METHODS: The authors studied the response of periodontal ligament cells to this pool of growth factors on cell proliferation, cell migration, secretion of several biomolecules, cell adhesion, and expression of α2 integrin. Cell proliferation and adhesion were evaluated by means of a fluorescence-based method. Cell migration was performed on culture inserts. The release of different biomolecules by periodontal ligament fibroblasts was quantified through enzyme-linked immunosorbent assay. The α2 integrin expression was assessed through Western blot. RESULTS: This autologous technology significantly stimulated cell proliferation, migration, adhesion, and synthesis of many growth factors from cells including vascular endothelial growth factor, thrombospondin 1, connective tissue growth factor, hepatocyte growth factor, and procollagen type I. The α2 integrin expression was lower in plasma rich in growth factor-treated cells compared to non-stimulated cells, although no statistically significant differences were observed. CONCLUSION: This plasma rich in growth factors exerts positive effects on periodontal ligament fibroblasts, which could be positive for periodontal regeneration.

HGF regulates the activation of TGF-ß1 in rat hepatocytes and hepatic stellate cells.

Hepatocyte growth factor (HGF) ameliorates experimental liver fibrosis through many mechanisms, including degradation of accumulated collagen and decreased expression of fibrotic genes. Investigating an upstream mechanism in which HGF could decrease many fibrotic effectors, we asked whether HGF regulates activation of the fibrotic cytokine transforming growth factor-beta 1 (TGF-ß1). Specifically, we tested whether HGF decreases the levels of active TGF-ß1, and whether such decrease depends on the predominantly hepatocyte-secreted protease plasmin, and whether it depends on the TGF-ß1 activator thrombospondin-1 (TSP-1). With hepatocyte monocultures, we found HGF-induced hepatocyte proliferation did increase total levels of plasmin, while decreasing gene expression of fibrotic markers (PAI-1, TGF-ß1, and TIMP-2). With in vitro models of fibrotic liver (HSC-T6 hepatic stellate cells, or co-cultures of HSC-T6 and hepatocytes), we found high levels of fibrosis-associated proteins such as TSP-1, active TGF-ß1, and Collagen I. HGF treatment on these fibrotic cultures stimulated plasmin levels; increased TSP-1 protein cleavage; and decreased the levels of active TGF-ß1 and Collagen I. When plasmin was blocked by the inhibitor aprotinin, HGF could no longer decrease TGF-ß1 activation and Collagen I. Meanwhile, the TSP-1-specific peptide inhibitor, LSKL, reduced TGF-ß1 to the same level as in the HGF-treated cultures; combining LSKL and HGF treatments caused no further decrease, suggesting that HGF affects the TSP-1 dependent pathway of TGF-ß1 activation. Therefore, HGF can decrease TGF-ß1 activation and TGF-ß1-dependent fibrotic markers, by stimulating hepatocytes to produce plasmin, and by antagonizing TSP-1-dependent activation of TGF-ß1.

Thrombospondin-1 and pigment epithelium-derived factor enhance responsiveness of KM12 colon tumor to metronomic cyclophosphamide but have disparate effects on tumor metastasis.

The anti-tumor activity, metronomic chemotherapy sensitization potential and metastatic effects of the endogenous angiogenesis inhibitors thrombospondin-1 and PEDF were investigated in KM12 colon adenocarcinoma xenografts. Thrombospondin-1 and PEDF decreased KM12 tumor microvessel density, increased macrophage infiltration, and improved responsiveness to metronomic cyclophosphamide (CPA) treatment, but did not activate the anti-tumor innate immunity that metronomic CPA induces in other tumor models. Moreover, thrombospondin-1, but not PEDF, significantly increased KM12 metastasis to the lung, while PEDF augmented the anti-metastatic activity of metronomic CPA. Thus, while thrombospondin-1 and PEDF both increase the KM12 tumor responsiveness to metronomic CPA, they have disparate effects on tumor metastasis.

Angiogenesis regulatory factors in the vitreous from patients with proliferative diabetic retinopathy.

We determined the levels of the endogenous angiogenesis inhibitors soluble vascular endothelial growth factor receptor-1 (sVEGFR-1), thrombospondin (TSP)-1 and TSP-2 in the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) and correlated their levels with clinical disease activity and the levels of vascular endothelial growth factor (VEGF). Vitreous samples from 30 PDR and 25 nondiabetic patients were studied by enzyme-linked immunosorbent assay. TSP-1 was not detected. VEGF and TSP-2 levels were significantly higher in PDR with active neovascularization compared with inactive PDR and nondiabetic patients (P < 0.001 for both comparisons). VEGF, sVEGFR-1 and TSP-2 levels were significantly higher in PDR with hemorrhage compared with PDR without hemorrhage and nondiabetic patients (P = 0.0063; 0.0144; <0.001, respectively). VEGF and sVEGFR-1 levels were significantly higher in PDR without traction retinal detachment (TRD) compared with PDR with TRD and nondiabetic patients (P = 0.038; 0.022, respectively). TSP-2 levels were significantly higher in PDR with TRD compared with PDR without TRD and nondiabetic patients (P < 0.001). There was a significant correlation between levels of VEGF and sVEGFR-1 (r = 0.427, P = 0.038). Our findings suggest that upregulation of sVEGFR-1 and TSP-2 may be a protective mechanism against progression of angiogenesis associated with PDR. TSP-2 might be associated with TRD.

Thrombospondin-1 inhibits osteogenic differentiation of human mesenchymal stem cells through latent TGF-ß activation.

Transforming growth factor-ß (TGF-ß) is a critical regulator of bone development and remodeling. TGF-ß must be activated from its latent form in order to signal. Thrombospondin-1 (TSP1) is a major regulator of latent TGF-ß activation and TSP1 control of TGF-ß activation is critical for regulation of TGF-ß activity in multiple diseases. Bone marrow-derived mesenchymal stem cells (MSCs) have osteogenic potential and they participate in bone remodeling in injury and in response to tumor metastasis. Since both TSP1 and TGF-ß inhibit osteoblast differentiation, we asked whether TSP1 blocks osteoblast differentiation of MSCs through its ability to stimulate TGF-ß activation. TSP1 added to human bone marrow-derived MSCs under growth conditions increases active TGF-ß. Cultured MSCs express TSP1 and both TSP1 expression and TGF-ß activity decrease during osteoblast differentiation. TSP1 and active TGF-ß block osteoblast differentiation of MSCs grown in osteogenic media as measured by decreased Runx2 and alkaline phosphatase expression. The inhibitory effect of TSP1 on osteoblast differentiation is due to its ability to activate latent TGF-ß, since a peptide which blocks TSP1 TGF-ß activation reduced TGF-ß activity and restored osteoblast differentiation as measured by increased Runx2 and alkaline phosphatase expression. Anti-TGF-ß neutralizing antibody also increased alkaline phosphatase expression in the presence of TSP1. These studies show that TSP1 regulated TGF-ß activity is a critical determinant of osteoblast differentiation.

High confidence and sensitivity four-dimensional fractionation for human plasma proteome analysis.

Reducing the complexity of plasma proteome through complex multidimensional fractionation protocols is critical for the detection of low abundance proteins that have the potential to be the most specific disease biomarkers. Therefore, we examined a four dimension profiling method, which includes low abundance protein enrichment, tryptic digestion and peptide fractionation by IEF, SCX and RP-LC. The application of peptide pI filtering as an additional criterion for the validation of the identifications allows to minimize the false discovery rate and to optimize the best settings of the protein identification database search engine. This sequential approach allows for the identification of low abundance proteins, such as angiogenin (10(-9) g/L), pigment epithelium growth factor (10(-8) g/L), hepatocyte growth factor activator (10(-7) g/L) and thrombospondin-1 (10(-6) g/L), having concentrations similar to those of many other growth factors and cytokines involved in disease pathophysiology.

Overexpression of thrombospondin-1 in stromal myofibroblasts is associated with tumor growth and nodal metastasis in gastric carcinoma.

BACKGROUND AND OBJECTIVES: The roles of thrombospondin-1 (THBS-1) in tumor growth and metastasis are complicated and its function as a cancer inhibitor or promoter remains controversial. This clinical study investigated the functional roles of THBS-1 in gastric carcinoma by examining the expression patterns of THBS-1 protein and mRNA levels during gastric cancer development. METHODS: Eighty-two gastric carcinomas were included in this study. THBS-1, α-smooth muscle actin, and CD34 proteins were localized by immunohistochemical staining, and the levels of THBS-1 mRNA were quantified by real-time polymerase chain reaction. RESULTS: THBS-1 mRNA expression in gastric carcinoma tissues was significantly higher than in adjacent non-cancerous stomach tissues (P = 0.03). Tumor THBS-1 mRNA expression level was significantly related to lymph node metastasis (P = 0.031), tumor size (P = 0.021) and patient age (P = 0.005). THBS-1 protein was mainly located in stromal myofibroblasts, and was undetectable in tumor cells. Myofibroblasts may be mainly derived from stromal fibroblasts in gastric cancer. The abundance of myofibroblasts was positively correlated with tumor growth and nodal metastasis in gastric carcinoma (P = 0.03, P = 0.0008, respectively). CONCLUSIONS: This clinical study revealed that overexpression of THBS-1 in stromal myofibroblasts is associated with tumor growth and nodal metastasis in gastric carcinoma. THBS-1 may activate latent transforming growth factor-ß1 to stimulate fibroblasts to differentiate into myofibroblasts, though further studies are needed to validate this hypothesis. These results suggest that THBS-1 and myofibroblasts may serve as novel targets for strategies aimed at protection against and treatment of gastric carcinoma.