Thrombospondin-1 (TSP1/THBS1) plays a major role in the pathophysiology of rheumatoid arthritis (RA); however, its interface with the cytokine network involved in RA has not been delineated. Correlations were performed between plasma levels of TSP1 and selected cytokines from blood samples collected from 20 patients affected by RA and 13 healthy donors (control). Plasma levels of TSP1 and tissue growth factor beta (TGFbeta) were determined by standard enzyme-linked immunosorbent assay, and cytokines were measured by protein profiling rolling-circle amplification (RCA). TSP1 circulating levels in plasma were found significantly increased in the RA patients when compared with control individuals (P = 0.039). The plasma levels of TGFbeta were also increased in the RA patients, which indicates a statistical trend. Cytokine levels of interleukin (IL)-4, IL-5, IL-12, chemokine CXC 10 (CXCL10/IP10), and chemokine CC 4 (CCL4)/MIP1beta were significantly increased in the RA patients when compared with the control group. In summary, this study demonstrates increased plasma levels of TSP1, which correlated with increased levels of proinflammatory cytokines in plasma of RA patients. More detailed research is required to explore the cytokine imprint yielded by this study and its interface with TSP1 and TGFbeta.

To date, there is no available targeted therapy for patients who are diagnosed with triple-negative breast cancers (TNBC). The aim of this study was to identify a new specific target for specific treatments. Frozen primary tumors were collected from 83 adjuvant therapy-naive TNBC patients. These samples were used for global proteome profiling by iTRAQ-OFFGEL-LC-MS/MS approach in two series: a training cohort (n = 42) and a test set (n = 41). Patients who remains free of local or distant metastasis for a minimum of 5 years after surgery were classified in the no-relapse group; the others were in the relapse group. OPLS and Kaplan-Meier analyses were performed to select candidate markers, which were validated by immunohistochemistry. Three proteins were identified in the training set and validated in the test set by Kaplan-Meier method and immunohistochemistry (IHC): TrpRS as a good prognostic markers and DP and TSP1 as bad prognostic markers. We propose the establishment of an IHC test to calculate the score of TrpRS, DP, and TSP1 in TNBC tumors to evaluate the degree of aggressiveness of the tumors. Finally, we propose that DP and TSP1 could provide therapeutic targets for specific treatments.

High-temperature requirement protein A1 (HTRA1) is a serine protease secreted by a number of tissues including retinal pigment epithelium (RPE). A promoter variant of the gene encoding HTRA1 is part of a mutant allele that causes increased HTRA1 expression and contributed to age-related macular degeneration (AMD) in genomewide association studies. AMD is characterized by pathological development of drusen, extracellular deposits of proteins and lipids on the basal side of RPE. The molecular pathogenesis of AMD is not well understood, and understanding dysregulation of the extracellular matrix may be key. We assess the high-risk genotype at 10q26 by proteomic comparison of protein levels of RPE cells with and without the mutation. We show HTRA1 protein level is increased in high-risk RPE cells along with several extracellular matrix proteins, including known HTRA1 cleavage targets LTBP-1 and clusterin. In addition, two novel targets of HTRA1 have been identified: EFEMP1, an extracellular matrix protein mutated in Doyne honeycomb retinal dystrophy, a genetic eye disease similar to AMD, and thrombospondin 1 (TSP1), an inhibitor of angiogenesis. Our data support the role of RPE extracellular deposition with potential effects in compromised barrier to neovascularization in exudative AMD.

CD47-binding sequences from the carboxyl-terminal domain of thrombospondin-1 (TSP1) are known to regulate activity of the alpha(v)beta(3) integrin (Gao, G., Lindberg, F. P., Dimitry, J. M., Brown, E. J., and Frazier, W. A. (1996) J. Cell Biol. 135, 533-544). Here we show that peptides from the type 1 repeats of TSP1 also stimulate alpha(v)beta(3) integrin function in melanoma cells. Addition of soluble peptide 246 (KRFKQDGGWSHWSPWSS) enhances spreading of A2058 melanoma cells on several alpha(v)beta(3) integrin ligands, including vitronectin, recombinant TSP1 fragments containing the Arg-Gly-Asp sequence, and native TSP1. This activity requires the Trp residues and is independent of CD36-binding sequences in the type 1 repeats. Recombinant type 1 repeats expressed as a glutathione S-transferase fusion protein also enhance spreading on vitronectin and TSP1. Activation of alpha(v)beta(3) integrin by the soluble peptide 246 stimulates organization of F-actin and increases tyrosine phosphorylation of focal adhesion kinase. In contrast, direct adhesion of melanoma cells on immobilized peptide 246 inhibits tyrosine phosphorylation of focal adhesion kinase. Stimulation of alpha(v)beta(3) integrin function by the type 1 repeat peptide differs from that induced by CD47-binding TSP1 peptides in that heparan sulfate proteoglycans are required and pertussis toxin does not inhibit the former activity. Thus, the type 1 repeats contain a second sequence of TSP1 that can enhance alpha(v)beta(3) integrin signaling, and these two sequences stimulate recognition of both vitronectin and TSP1 by the alpha(v)beta(3) integrin.

Thrombospondin-1 and -2 (TSP1 and TSP2) are multifunctional, multimodular extracellular matrix proteins encoded by separate genes. We compared the distributions of TSP1 and TSP2 in mouse embryos (day 10 and later) by immunohistochemistry. TSP1 was detected on day 10 in the heart and intestinal epithelium, on day 11 in megakaryocytes, and on day 14 in the lung. TSP2 was not detected until day 14, with strongest staining in mesenchymal condensation that gives rise to cartilage and bone. The distribution of TSP2 was different from but overlapped with the distribution of TSP1. TSP1 was found in cartilage proper with diminished staining around chondrocytes undergoing differentiation and hypertrophy, whereas TSP2 was restricted to the matrix surrounding chondrocytes of the growth zone cartilage. TSP2 and TSP1 were both expressed in centers of intramembranous ossification that form the skull bones, in reticular dermis, on the apical surface of nasal epithelium, in skeletal muscle, and in the sheath surrounding vibrissae. Areas of exclusive staining for TSP2 included the perichondrium surrounding the cartilage of the nasal cavities, developing bone of the lower mandible, and adrenal gland. The distinct localizations of TSP1 and TSP2 indicate that the two proteins have specific functions during mouse embryogenesis.

The initiation of angiogenesis can mark the transition from tumor dormancy to active growth and recurrence. Mechanisms that regulate recurrence in human cancers are poorly understood, in part because of the absence of relevant models. The induction of ARHI (DIRAS3) induces dormancy and autophagy in human ovarian cancer xenografts but produces autophagic cell death in culture. The addition of VEGF to cultures maintains the viability of dormant autophagic cancer cells, thereby permitting active growth when ARHI is downregulated, which mimics the "recurrence" of growth in xenografts. Two inducible ovarian cancer cell lines, SKOv3-ARHI and Hey-ARHI, were used. The expression level of angiogenesis factors was evaluated by real-time PCR, immunohistochemistry, immunocytochemistry and western blot; their epigenetic regulation was measured by bisulfite sequencing and chromatin immunoprecipitation. Six of the 15 angiogenesis factors were upregulated in dormant cancer cells (tissue inhibitor of metalloproteinases-3, TIMP3; thrombospondin-1, TSP1; angiopoietin-1; angiopoietin-2; angiopoietin-4; E-cadherin, CDH1). We found that TIMP3 and CDH1 expression was regulated epigenetically and was related inversely to the DNA methylation of their promoters in cell cultures and in xenografts. Increased H3K9 acetylation was associated with higher TIMP3 expression in dormant SKOv3-ARHI cells, while decreased H3K27me3 resulted in the upregulation of TIMP3 in dormant Hey-ARHI cells. Elevated CDH1 expression during dormancy was associated with an increase in both H3K4me3 and H3K9Ac in two cell lines. CpG demethylating agents and/or histone deacetylase inhibitors inhibited the re-growth of dormant cancer cells, which was associated with the re-expression of anti-angiogenic genes. The expression of the anti-angiogenic genes TIMP3 and CDH1 is elevated during dormancy and is reduced during the transition to active growth by changes in DNA methylation and histone modification.

OBJECTIVE

Despite advances in stent design, in-stent restenosis (ISR) remains a significant clinical problem. All implant metals exhibit corrosion, which results in release of metal ions. Stainless steel (SS), a metal alloy widely used in stents, releases ions to the vessel wall and induces reactive oxygen species, inflammation and fibroproliferative responses. The molecular mechanisms are unknown. TGF-beta is known to be involved in the fibroproliferative responses of vascular smooth muscle cells (VSMCs) in restenosis, and TGF-beta antagonists attenuate ISR. We hypothesized that SS ions induce the latent TGF-beta activator, thrombospondin-1 (TSP1), through altered oxidative signaling to stimulate increased TGF-beta activation and VSMC phenotype change.

METHODS

VSMCs were treated with SS metal ion cocktails, and morphology, TSP1, extracellular matrix production, desmin and TGF-beta activity were assessed by immunoblotting.

RESULTS

SS ions stimulate the synthetic phenotype, increased TGF-beta activity, TSP1, increased extracellular matrix and downregulation of desmin in VSMCs. Furthermore, SS ions increase hydrogen peroxide and decrease cGMP-dependent protein kinase (PKG) signaling, a known repressor of TSP1 transcription. Catalase blocks SS ion attenuation of PKG signaling and increased TSP1 expression.

CONCLUSIONS

These data suggest that ions from stent alloy corrosion contribute to ISR through stimulation of TSP1-dependent TGF-beta activation.

Modulation by thyroid hormone and hormone analogs of angiogenesis in the heart after experimental infarction, and in other organs, has been appreciated for decades. Description of a plasma membrane receptor for thyroid hormone on the extracellular domain of integrin αvβ3 on endothelial cells has revealed the complexity of the nongenomic regulation of angiogenesis by the hormone. From αvβ3, the hormone directs transcription of specific vascular growth factor genes, regulates growth factor receptor/growth factor interactions and stimulates endothelial cell migration to a vitronectin cue; these actions are implicated experimentally in tumor-relevant angiogenesis and angioproliferative pulmonary hypertension. Derived from L-thyroxine (T4), tetraiodothyroacetic acid (tetrac) can be covalently bound to a polymer and as Nanotetrac acts exclusively at the hormone receptor on αvβ3 to block actions of T4 and 3,5,3'-triiodo-L-thyronine (T3) on angiogenesis. Other antiangiogenic actions of Nanotetrac include disruption of crosstalk between integrin αvβ3 and adjacent cell surface vascular growth factor receptors, resulting in disordered vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF; FGF2) actions at their respective plasma membrane receptors. From αvβ3, Nanotetrac also downregulates expression of VEGFA and epidermal growth factor receptor (EGFR) genes, upregulates transcription of the angiogenesis suppressor gene, thrombospondin 1 (THBS1; TSP1) and decreases cellular abundance of Ang-2 protein and matrix metalloproteinase-9. Existence of this receptor provides new insights into the multiple mechanisms by which thyroid hormone and hormone analogs may regulate angiogenesis at the molecular level. The receptor also offers pharmacological opportunities for interruption of pathological angiogenesis via integrin αvβ3.

Interactions of heparin with intact human thrombospondin-1 (TSP1) and with two heparin-binding fragments of TSP1 were characterized using chemically modified heparins, a vascular heparan sulfate proteoglycan, and a series of heparin oligosaccharides prepared by partial deaminative cleavage. The avidity of TSP1 binding increased with oligosaccharide size, with plateaus at 4 to 6 and at 8 to 10 monosaccharide units. The dependence on oligosaccharide size for binding to the recombinant amino-terminal heparin-binding domain of TSP1 was the same as that of the intact TSP1 molecule but differed from that of a synthetic heparin-binding peptide from the type 1 repeats, suggesting that the interaction between intact TSP1 and heparin is primarily mediated by the amino-terminal domain. Based on activities of chemically modified heparins, binding to TSP1 depended primarily on 2-N- and 6-O-sulfation of glucosamine and to a lesser degree on 2,3-O-sulfation and the carboxyl residues of the uronic acids. In contrast, all of these modifications were required for binding of heparin to the type 1 repeat peptides. Affinity purification of heparin octasaccharides on immobilized TSP1 type 1 repeat peptides revealed a preference for oligosaccharides containing the disaccharide sequence IdoA(2-OSO(3))alpha1-4-GlcNS(6-OSO(3)). Binding of these oligosaccharides to the peptide required the Trp residues. These data demonstrate that the heparin-binding specificities of intact TSP1 and peptides from the type 1 repeats overlap with that of basic fibroblast growth factor (FGF2) and are consistent with the ability of these TSP1-derived molecules to inhibit FGF2-stimulated angiogenesis.

Transforming growth factor-β (TGF-β) is a critical regulator of bone development and remodeling. TGF-β must be activated from its latent form in order to signal. Thrombospondin-1 (TSP1) is a major regulator of latent TGF-β activation and TSP1 control of TGF-β activation is critical for regulation of TGF-β 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-β inhibit osteoblast differentiation, we asked whether TSP1 blocks osteoblast differentiation of MSCs through its ability to stimulate TGF-β activation. TSP1 added to human bone marrow-derived MSCs under growth conditions increases active TGF-β. Cultured MSCs express TSP1 and both TSP1 expression and TGF-β activity decrease during osteoblast differentiation. TSP1 and active TGF-β 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-β, since a peptide which blocks TSP1 TGF-β activation reduced TGF-β activity and restored osteoblast differentiation as measured by increased Runx2 and alkaline phosphatase expression. Anti-TGF-β neutralizing antibody also increased alkaline phosphatase expression in the presence of TSP1. These studies show that TSP1 regulated TGF-β activity is a critical determinant of osteoblast differentiation.

In this report, we use a proteomic strategy to identify glycoproteins on the surface of exosomes derived from myeloid-derived suppressor cells (MDSCs), and then test if selected glycoproteins contribute to exosome-mediated chemotaxis and migration of MDSCs. We report successful modification of a surface chemistry method for use with exosomes and identify 21 surface N-glycoproteins on exosomes released by mouse mammary carcinoma-induced MDSCs. These glycoprotein identities and functionalities are compared with 93 N-linked glycoproteins identified on the surface of the parental cells. As with the lysate proteomes examined previously, the exosome surface N-glycoproteins are primarily a subset of the glycoproteins on the surface of the suppressor cells that released them, with related functions and related potential as therapeutic targets. The "don't eat me" molecule CD47 and its binding partners thrombospondin-1 (TSP1) and signal regulatory protein α (SIRPα) were among the surface N-glycoproteins detected. Functional bioassays using antibodies to these three molecules demonstrated that CD47, TSP1, and to a lesser extent SIRPα facilitate exosome-mediated MDSC chemotaxis and migration.

Recognition and invasion of host cells is a key step in the life-cycle of all apicomplexan parasites. The thrombospondin-related anonymous protein (TRAP) of Plasmodium sporozoites is directly involved in both processes and shares conserved adhesive domains with micronemal transmembrane proteins of other apicomplexans. Here, we report the cloning and characterization of a Babesia bovis TRAP homologue (BbTRAP). It was predicted to be a type 1 transmembrane protein containing a von Willebrand Factor A domain (vWFA), a thrombospondin type 1 domain (TSP1), a conserved transmembrane region and a conserved cytoplasmic C-terminus, thus resembling the domain arrangement of Plasmodium TRAP. In contrast to Plasmodium TRAP, BbTRAP was shown to be present during the asexual erythrocytic cycle, being located mainly at the apical side of merozoites. Polyclonal rabbit antisera directed against synthetic peptides derived from the TSP1 domain or the C-terminal end of the ectodomain were shown to inhibit erythrocyte invasion in vitro. Both antisera recognized a 75 kDa protein in merozoite extracts as well as in a protein fraction that was secreted into the extracellular milieu during in vitro invasion of erythrocytes.

Thrombospondin 1 (TSP1) is an angiogenesis inhibitor that decreases tumor growth. We now report that TSP1 directly inhibits the proliferation of human melanoma cells. TSP1, peptides, and a recombinant fragment from the type I repeats, but not peptides that bind CD36 or CD47, inhibit the proliferation of A2058 melanoma cells. In contrast, chemotaxis is mediated by peptides or recombinant fragments from the procollagen, type I, type II, and cell-binding domains. The antiproliferative activity of TSP1 is mediated by a different signal transduction pathway than those mediating motility responses to the same protein. Activators of protein kinase A and protein kinase C inhibit chemotaxis but not the antiproliferative activity of TSP1, whereas the antiproliferative activity is reversed by inhibiting the tyrosine kinase or phosphatase activities. TSP1-mediated chemotaxis is partially dependent on a pertussis toxin (PT)-sensitive G-binding protein, whereas haptotaxis is not. Chemotaxis stimulated by the procollagen domain and the CD47-binding sequences from the COOH-terminal domain are also sensitive to PT, but responses to the type I and type III domains are not sensitive to PT. Residual chemotaxis to TSP1 in the presence of PT may therefore be mediated by the activities of the type I or type III repeats. Thus, TSP1 elicits several intracellular signals in melanoma cells that result from interactions with several domains of this protein and differentially affect growth and motility.

OBJECTIVE

Platelets abundantly express the membrane receptor CD36 and store its ligand thrombospondin-1 (TSP1) in the α-granules. We investigated whether released TSP1 can support platelet adhesion and thrombus formation via interaction with CD36.

RESULTS

Mouse platelets deficient in CD36 showed reduced adhesion to TSP1 and subsequent phosphatidylserine expression. Deficiency in either CD36 or TSP1 resulted in markedly increased dissolution of thrombi formed on collagen, although thrombus buildup was unchanged. In mesenteric vessels in vivo, deficiency in CD36 prolonged the time to occlusion and enhanced embolization, which was in agreement with earlier observations in TSP1-deficient mice. Thrombi formed using wild-type blood stained positively for secreted TSP1. Releasate from wild-type but not from TSP1-deficient platelets enhanced platelet activation, phosphatidylserine expression, and thrombus formation on collagen. The enhancement was dependent on CD36 because it was without effect on thrombus formation by CD36-deficient platelets.

CONCLUSIONS

These results demonstrate an anchoring role of platelet-released TSP1 via CD36 in platelet adhesion and collagen-dependent thrombus stabilization. Thus, the TSP1-CD36 tandem is another platelet ligand-receptor axis contributing to the maintenance of a stable thrombus.

OBJECTIVE

There is a need for biomarkers that may help in selecting the most effective anticancer treatments for each patient. We have investigated the prognostic value of a set of angiogenesis, inflammation and coagulation markers in patients treated for advanced non-small cell lung cancer.

METHODS

Peripheral blood samples were obtained from 60 patients before first line platinum-based chemotherapy ± bevacizumab, and after the third cycle of treatment. Blood samples from 60 healthy volunteers were also obtained as controls. Angiogenesis, inflammation and coagulation markers vascular endothelial growth factor (VEGF), their soluble receptors 1 (VEGFR1) and 2 (VEGFR2), thrombospondin-1 (TSP-1), interleukin-6 (IL6), sialic acid (SA) and tissue factor (TF) were quantified by ELISA.

RESULTS

Except for TSP-1, pre- and post-treatment levels of all markers were higher in patients than in controls (p < 0.05). There was a positive and significant correlation between VEGF and VEGFR2 before treatment. VEGF also correlated with inflammatory markers IL-6 and SA. Moreover, there was a positive and significant correlation between levels of VEGFR1 and TF. Decreased levels of TSP-1 and increased levels of VEGF were associated with shorter survival. Bevacizumab significantly modified angiogenesis parameters and caused a decrease of VEGF and an increase of TSP-1.

CONCLUSIONS

Angiogenesis, inflammation and coagulation markers were increased in NSCLC patients. Increased levels of VEGF and low levels of TSP-1 correlated with a poor prognosis.

OBJECTIVE

Potential predictive/prognostic angiogenic markers were prospectively examined in a phase II trial of bevacizumab in epithelial ovarian cancer (EOC)/primary peritoneal cancer (PPC).

METHODS

Recurrent/persistent EOC/PPC patients were treated with bevacizumab (15 mg/kg IV q21days) until disease progression. Validated-immunohistochemistry (IHC) assays were performed on pre-cycle 1/4 tumor biopsies for CD31-microvessel density (MVD), VEGF-histoscore (HS), p53-HS, and TSP1 image analysis score (IA). Pre-cycle 1/4 serum and plasma VEGF were quantified using a validated-ELISA.

RESULTS

CD31-MVD and serum VEGF, evaluated pre-cycle 1 in 41/61 and 51/61 eligible patients, respectively, did not appear to be correlated. High CD31-MVD, categorized at the median, appeared to be associated with tumor response, a 13-month shorter median survival, and an increased risk of death (unadjusted hazard ratio [HR] = 2.2, 95% confidence interval [CI] = 1.067-4.467). In addition, each standard deviation (SD) increase in CD31-MVD appeared to be associated with worse survival in unadjusted and adjusted analyses. IHC and plasma biomarkers did not change with bevacizumab treatment except for serum VEGF, which appeared to decrease during bevacizumab treatment. This decrease was not associated with response. High pre-cycle 1 serum VEGF, categorized at the median, was associated with 22-month shorter median survival and an increased risk of death (unadjusted HR = 2.7, 95% CI = 1.369-5.191). Categorized p53 appeared to be associated with unadjusted survival and each SD increase in TSP1-IA appeared to be associated with a decreased risk of progression in unadjusted and adjusted analyses.

CONCLUSIONS

Despite the limitations in sample size and exploratory nature of the study, angiogenic markers in tumor and serum may provide prognostic value in recurrent/persistent EOC/PPC, and are being prospectively evaluated in the GOG phase III trial of carboplatin, paclitaxel and bevacizumab/placebo in previously untreated EOC/PPC.

Overexpression of a novel oncogene MCT-1 (multiple copies in a T cell malignancy) causes malignant transformation of murine fibroblasts. To establish its role in the pathogenesis of breast cancer in humans, we generated stable transfectants of MCF7 breast cancer cells negative for endogenous MCT-1 (MCF7-MCT-1). Overexpression of MCT-1 in these cells resulted in a slight elevation of estrogen receptor-alpha, and higher rates of DNA synthesis and growth in response to estradiol compared with the empty vector control (MCF7-EV). The pure antiestrogen fulvestrant inhibited the estradiol-stimulated proliferation of MCF7-MCT-1 cells. The MCF7-MCT-1 clones showed increased invasiveness in the presence of 50% serum compared with the MCF7-EV. In a tumor xenograft model, MCT-1-overexpressing cells showed higher take rates and formed significantly larger tumors than MCF7-EV controls. When we examined angiogenic phenotype and molecular mediators of angiogenesis in MCF7-MCT-1 tumors in vivo, we found greater microvascular density and lower apoptosis in the MCF7-MCT-1 tumors compared with MCF7-EV controls accompanied by a dramatic decline in the levels of angiogenesis inhibitor, thrombospondin-1 (TSP1). In vitro, blocking TSP1 in the medium conditioned by MCT-1-negative cells restored its angiogenic potential to that of the MCF7-MCT-1 cells. Conversely, despite an increase in mRNA encoding vascular endothelial growth factor upon MCT-1 overexpression, vascular endothelial growth factor protein levels have not been notably altered. Taken together, our results suggest that MCT-1 may contribute to the pathogenesis and progression of human breast cancer via at least two routes: promotion of angiogenesis through the decline of TSP1 and inhibition of apoptosis.

Although it has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine, the mechanisms by which T. spiralis infective larvae recognize and invade the intestinal epithelial cells (IECs) are unknown. It is speculated that the molecular interactions between the parasite and host enterocytes may mediate the recognition and invasion of IECs by T. spiralis. However, no Trichinella proteins that interact with the enterocytes have been identified previously. The aim of this study was to identify Trichinella proteins that bind to IECs by screening a T7 phage display cDNA library constructed using messenger RNA from T. spiralis intestinal infective larvae. Following five rounds of biopanning, sequencing, and bioinformatics analysis, ten T. spiralis proteins (<em>Tsp1</em>-<em>Tsp1</em>0) with significant binding to normal mouse IECs were identified. The results of the protein classification showed that six proteins (<em>Tsp1</em>, calcium-transporting ATPase 2 protein; Tsp4, ovochymase-1; Tsp6, T-complex protein 1 subunit eta; Tsp7, glycosyl hydrolase family 47; Tsp8, DNA replication licensing factor MCM3; and <em>Tsp1</em>0, nudix hydrolase) of these T. spiralis proteins were annotated with putative molecular functions. Out of the six proteins, five have catalytic activity, four have binding activity, and one has transporter activity. Anti-<em>Tsp1</em>0 antibodies prevented the in vitro partial invasion of IECs by infective larvae and the mice immunized with the recombinant phage T7-<em>Tsp1</em>0 showed a 62.8 % reduction in adult worms following challenge with T. spiralis muscle larvae. Although their biological functions are not yet fully known, these proteins might be related to the larval invasion of host enterocytes. Future experiments will be necessary to ascertain whether these proteins play important roles in the recognition and invasion of host enterocytes. The construction and biopanning of Trichinella phage display libraries provide a novel approach for searching for candidate genes that are related to invasion and for identifying protein interactions between parasite and host.

Crohn's disease and ulcerative colitis are inflammatory bowel diseases (IBD) quite common in the United States and other Western countries. Patients suffering IBD are at greater risk of developing colorectal adenocarcinoma than the general population. Both, the adenoma-carcinoma and the inflammation-carcinogenesis processes are characterized by active angiogenesis. Recent studies also have shown that anti-angiogenesis might be a novel therapeutic approach for IBD. Thrombospondin 1 (TSP1) is an extracellular protein well known for its anti-angiogenic properties. TSP1 also has key functions in inflammation, which is assumed to be the primary cause for carcinogenesis in IBD. This review is focused on the role of TSP1 in colorectal carcinogenesis. The therapeutic effects of TSP derived-peptides on inhibiting the inflammationcarcinogenesis progression are also discussed.

OBJECTIVE

We tested the hypothesis that the matricellular protein thrombospondin-1 (TSP1), through binding to and activation of the cell receptor CD47, inhibits basal and thermal-mediated cutaneous blood flow.

BACKGROUND

Abnormal and decreased cutaneous blood flow in response to temperature changes or vasoactive agents is a feature of cardiovascular disease and aging. The reasons for decreased cutaneous blood flow remain incompletely understood. Furthermore, a role for matricellular proteins in the regulation skin blood flow has never been proposed.

METHODS

C57BL/6 wild type, TSP1-null, and CD47-null 12- and 72-week-old male mice underwent analysis of skin blood flow (SkBF) via laser Doppler in response to thermal stress and vasoactive challenge.

RESULTS

Young and aged TSP1- and CD47-null mice displayed enhanced basal and thermal sensitive SkBF changes compared with age-matched wild type controls. Nitric oxide-mediated increases in SkBF were also greater in null mice. TSP1 and CD47 were expressed in skin from young wild type mice, and both were significantly upregulated in aged animals. Tissue 3',5'-cyclic guanosine monophosphate, a potent vasodilator, was greater in skin samples from null mice compared with wild type regardless of age. Finally, treating wild type animals with a CD47 monoclonal antibody that inhibits TSP1 activation of CD47 enhanced SkBF in both young and aged animals.

CONCLUSIONS

These results suggest that secreted TSP1, via its cognate receptor CD47, acutely modulates SkBF. These data further support therapeutically targeting CD47 to mitigate age-associated loss of SkBF and maximize wound healing.

OBJECTIVE

Spontaneous intracerebral hemorrhage (ICH) is among the most intractable forms of stroke. Angiogenesis, an orchestrated balance between proangiogenic and antiangiogenic factors, is a fundamental process to brain development and repair by new blood vessel formation from preexisting ones and can be induced by ICH. Thrombospondin (TSP)–1 and TSP-2 are naturally occurring antiangiogenic factors. The aim of this study was to observe their expression in rat brains with ICH.

METHODS

Intracerebral hemorrhage was induced in adult male Sprague-Dawley rats by stereotactic injection of collagenase VII or autologous blood into the right globus pallidus. The expression of TSP-1 and -2 was evaluated by immunohistochemistry and quantitative real-time reverse transcription–polymerase chain reaction analysis.

RESULTS

After the induction of ICH, some TSP1- or TSP2-immunoreactive microvessels resided around the hematoma for ~ 7 days and extended into a clot thereafter. Cerebral endothelial cells expressed the TSPs. The expression of TSP-1 and TSP-2 mRNA peaked at 4 and 14 days after collagenase-induced ICH, respectively.

CONCLUSIONS

Findings in this study suggest that ICH can alter the expression of TSP-1 and TSP-2, which may be involved in modulating angiogenesis in brains following ICH.

BACKGROUND

The kelch repeat protein muskelin mediates cytoskeletal responses to the extracellular matrix protein thrombospondin 1, (TSP1), that is known to promote synaptogenesis in the central nervous system (CNS). Muskelin displays intracellular localization and affects cytoskeletal organization in adherent cells. Muskelin is expressed in adult brain and has been reported to bind the Cdk5 activator p39, which also facilitates the formation of functional synapses. Since little is known about muskelin in neuronal tissues, we here analysed the tissue distribution of muskelin in rodent brain and analysed its subcellular localization using cultured neurons from multiple life stages.

RESULTS

Our data show that muskelin transcripts and polypeptides are expressed throughout the central nervous system with significantly high levels in hippocampus and cerebellum, a finding that resembles the tissue distribution of p39. At the subcellular level, muskelin is found in the soma, in neurite projections and the nucleus with a punctate distribution in both axons and dendrites. Immunostaining and synaptosome preparations identify partial localization of muskelin at synaptic sites. Differential centrifugation further reveals muskelin in membrane-enriched, rather than cytosolic fractions.

CONCLUSIONS

Our results suggest that muskelin represents a multifunctional protein associated with membranes and/or large protein complexes in most neurons of the central nervous system. These data are in conclusion with distinct roles of muskelin's functional interaction partners.

Vascular endothelial cell (EC)-produced plasma von Willebrand factor (vWF) plays a critical role in primary hemostasis through its action of anchoring platelets onto the injured denuded subendothelial matrices under high shear stress. Unusually large vWF multimers (UL-vWFMs), present in plasma immediately after release from ECs, are most biologically active, but they are soon cleaved and degraded into smaller vWFMs by a specific plasma protease, termed vWF-cleaving protease (vWF-CPase), in normal circulation. Recent studies on the relationship between UL-vWFMs and vWF-CPase, together with its autoantibody (inhibitor) have brought about a clear discrimination between thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Furthermore, a congenital deficiency of this enzyme activity has been shown to cause Upshaw-Schulman syndrome, a complex constitutional bleeding diathesis. Successful purification of vWF-CPase revealed that this enzyme is composed of a single polypeptide with a molecular mass of approximately 190 kd, and its complementary DNA cloning unambiguously indicated that it is uniquely produced in the liver and its gene is located on chromosome 9q34. The messenger RNA of vWF-CPase had a span of 4.6 kb, and its enzyme was designated ADAMTS 13. The predicted complete amino acid sequence of this enzyme consisted of 1427 residues, including a signal peptide, a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin-1 repeat (TSP1), a cysteine-rich domain, an ADAMTS spacer, 7 additional TSP1 repeats, and 2 CUB domains.

The mechanism by which proteolytic events translate into biological responses is not well understood. To explore the link of pericellular proteolysis to events relevant to capillary sprouting within the inflammatory context, we aimed at the identification of the collection of substrates of the protease MT1-MMP in endothelial tip cells induced by inflammatory stimuli. We applied quantitative proteomics to endothelial cells (ECs) derived from wild-type and MT1-MMP-null mice to identify the substrate repertoire of this protease in TNF-α-activated ECs. Bioinformatics analysis revealed a combinatorial MT1-MMP proteolytic program, in which combined rather than single substrate processing would determine biological decisions by activated ECs, including chemotaxis, cell motility and adhesion, and vasculature development. MT1-MMP-deficient ECs inefficiently processed several of these substrates (TSP1, CYR61, NID1, and SEM3C), validating the model. This novel concept of MT1-MMP-driven combinatorial proteolysis in angiogenesis might be extendable to proteolytic actions in other cellular contexts.