The N700S polymorphism of thrombospondin-1 (TSP-1) has been identified as a potential genetic risk factor for myocardial infarction (MI). In a large case-control study of 1425 individuals who survived a myocardial infarction prior to age 45, the N700S polymorphism was a significant risk factor for myocardial infarction in both homozygous (odds ratio [OR] 1.9, 95% confidence interval [CI] 1.1-3.3, P = .01) and heterozygous carriers of the S700 allele (OR 1.4, 95% CI 1.1-3.3, P = .01). TSP-1 has been shown to reduce von Willebrand factor (VWF) multimer size, and the domain responsible for VWF-reducing activity has been localized to the calcium-binding C-terminal sequence. As the N700S polymorphism was previously shown to alter the function of this domain, we investigated whether the altered VWF-reducing activity of TSP-1 underlies the observed prothrombotic phenotype. The TSP1 N700S polymorphism did not influence VWF multimer size in patients homozygous for either allele nor was there a significant reduction of VWF multimer size following incubation with recombinant N700S fragments or platelet-derived TSP-1.

We examined the effects of thrombospondin-1 (TSP1) and thrombospondin-2 (TSP2) on the uptake of tritiated thymidine by bovine aortic endothelial (BAE) cells in response to two growth factors, basic fibroblast growth factor (bFGF) and lysophosphatidic acid (LPA). bFGF and LPA stimulate cell proliferation through distinct receptors that have convergent signaling pathways. The doses of LPA that trigger proliferation of BAE cells, which have not been reported previously, were 1 to 30 micromol/L, as opposed to the 5 to 100 micromol/L concentrations required to stimulate proliferation of human foreskin fibroblasts. Baseline mitogenic activity and activity stimulated by either bFGF or LPA on BAE cells was inhibited by human TSP1 purified from platelets or a recombinant source with a similar dose response. These results demonstrate that the anti-proliferative effect of platelet TSP1 is not caused by contaminants from the stimulated platelet. Recombinant mouse TSP2 inhibited BAE cell proliferation in response to LPA in a dose range similar to that of TSP1. Inasmuch as TSP2 does not activate latent TGFbeta1 (Schultz-Cherry et al., J Biol Chem 1995;270: 7304), these results show that inhibition of angiogenesis by TSPs is not related to control of activation of TGFbeta. Together, these studies suggest that structural motifs common to TSP1 and TSP2 inhibit endothelial cell proliferation. Furthermore, TSPs inhibit cell proliferation stimulated by two growth factor receptors that act through distinct signaling pathways.

OBJECTIVE

Thrombospondin-1 (TSP1) is described as a positive regulator of vascular smooth muscle growth in cell culture. However, insight into the in vivo effects of TSP1 on smooth muscle cell (SMC) function is lacking.

RESULTS

We analyzed wild-type (WT) and TSP1-deficient (Tsp1-/-) mice in a carotid artery ligation model, in which neointimal lesions form without overt mechanical damage to the endothelium. On ligation, the expression of TSP1 increased strongly in the matrix of neointima and adventitia. In the early phase after ligation (day 3 to 7), activation, proliferation, and migration of medial SMCs were delayed and impaired in Tsp1-/- mice, in parallel with defective upregulation of metalloproteinase (MMP)-2 activity. As a result, Tsp1-/- arteries developed smaller neointimal lesions, a thicker media but comparably attenuated patency as in WT arteries, 28 days after ligation. Furthermore, medial and neointimal SMCs in Tsp1-/- mice produced more collagen, more osteopontin, and displayed weaker smooth muscle actin staining than WT SMCs, indicative of a modified SMC phenotype in Tsp1-/- mice.

CONCLUSIONS

Arterial SMC activation in the absence of TSP1 is delayed and dysregulated, reducing neointima formation, on mild vascular injury.

Counteradhesive proteins are a group of genetically and structurally distinct multidomain proteins that have been grouped together for their ability to inhibit cell-substrate interactions. Three counteradhesive proteins that influence endothelial cell behavior include thrombospondin (TSP)1, (SPARC) (Secreted Protein Acidic and Rich in Cysteine), also known as osteonectin, and tenascin. More recently, these proteins have been shown to regulate not only cell-matrix interactions but cell-cell interactions as well. TSP1 increases tyrosine phosphorylation of components of the cell-cell adherens junctions or zonula adherens (ZA) and opens the paracellular pathway in human lung microvascular endothelia. The epidermal growth factor (EGF)-repeats of TSP1 activate the (EGF) receptor (EGFR) and ErbB2, and these two receptor protein tyrosine kinases (PTK)s participate in ZA protein tyrosine phosphorylation and barrier disruption in response to the TSP1 stimulus. For the barrier response to TSP1, EGFR/ErbB2 activation is necessary but insufficient. Protein tyrosine phosphatase (PTP)mu counter-regulates phosphorylation of selected tyrosine residues within the cytoplasmic domain of EGFR. Although tenascin, like TSP1, also contains EGF-like repeats and is known to activate EGFR, whether it also opens the paracellular pathway is unknown. In addition to TSP1, tenascin, and the other TSP family members, there are numerous other proteins that also contain EGF-like repeats and participate in hemostasis, wound healing, and tissue remodeling. EGFR not only responds to direct binding of EGF motif-containing ligands but can also be transactivated by a wide range of diverse stimuli. In fact, several established mediators of increased vascular permeability and/or lung injury, including thrombin, tumor necrosis factor-alpha, platelet-activating factor, bradykinin, angiopoietin, and H(2)O(2), transactivate EGFR. It is conceivable that EGFR serves a pivotal signaling role in a final common pathway for the pulmonary response to selected injurious stimuli. SPARC/Osteonectin also increases tyrosine phosphorylation of ZA proteins and opens the endothelial paracellular pathway in a PTK-dependent manner. The expression of the counteradhesive proteins is increased in response to a wide range of injurious stimuli. It is likely that these same molecules participate in the host response to acute lung injury and are operative during the barrier response within the pulmonary microvasculature.

The extracellular matrix glycoprotein thrombospondin-1 (TSP1) inhibits angiogenesis, endothelial cell growth, motility and adhesion. Peptides from the type I repeats of TSP1 mimic the adhesive and growth inhibitory activities of the intact protein and specifically interact with heparin and transforming growth factor-beta (TGF beta). To define the structural basis for the antiangiogenic activities of these peptides, we prepared analogs of the TSP1 peptide KRFKQDGGWSHWSPWSSC. L-forward, L-reverse, and D-reverse (retro-inverso) analogs displayed identical activities for binding to heparin, demonstrating a lack of stereospecificity for heparin binding. The L-reverse and D-reverse peptides, however, had somewhat decreased abilities to activate latent TGF beta. Conjugation of the forward peptides through a C-terminal thioether and the reverse peptides through an N-terminal thioether to polysucrose abolished the adhesive activity of the peptides and enhanced their antiproliferative activities for endothelial and breast carcinoma cells stimulated by fibroblast growth factor-2. Their antiproliferative activities were independent of latent TGF beta activation, because substitution of an Ala residue for the essential Phe residue in the TSP1 type-1 repeat peptide increased their potency for inhibiting TSP1 binding to heparin and for inhibiting endothelial cell proliferation. Although the conjugated peptides were inactive in vivo, an unconjugated retro-inverso analog of the native TSP peptide inhibited breast tumor growth in a mouse xenograft model. Thus, these TSP-derived peptide analogs antagonize endothelial growth through their heparin-binding activity rather than through activation of latent TGF beta or increasing cell adhesion. These stable analogs may therefore be useful as therapeutic inhibitors of angiogenesis stimulated by fibroblast growth factor-2.

Several studies have supported the hypothesis that adrenocortical tumor formation is the result of a multistep process. The angiogenic switch has been proposed to be a key step in tumor progression from adenoma to carcinoma. In this study we measured the cytosolic concentrations of three proteins involved in angiogenesis [namely platelet-derived endothelial cell growth factor vascular endothelial cell growth factor A (VEGF-A), and thrombospondin-1 (TSP1)] in a series of 43 human sporadic adrenocortical tumors. The tumors were classified as adenomas (n = 18), transitional tumors (n = 12), or carcinomas (n = 13) according to the histological criteria defined by Weiss. Platelet-derived endothelial cell growth factor/thymidine phosphorylase levels were not significantly different among these three groups. One hundred percent of the adenomas and 73% of the transitional tumors showed VEGF-A concentrations under the threshold value of 107 ng/g protein, whereas 75% of the carcinomas had VEGF-A concentrations above this threshold value. Similarly, 89% of the adenomas showed TSP1 concentrations above the threshold value of 57 microg/g protein, whereas only 25% of the carcinomas and 33% of the transitional tumor samples did so. Insulin-like growth factor II overexpression, a common genetic alteration of adrenocortical carcinomas, was significantly correlated with higher VEGF-A and lower TSP1 concentrations. The tumors from the 6 patients with tumor recurrence after surgical ablation showed significantly higher VEGF-A values than the carcinomas and the transitional tumors from patients that did not relapse. Taken together, these data suggest that a decrease in TSP1 expression is an event that precedes an increase in VEGF-A expression during adrenocortical tumor progression. The population of premalignant tumors with low TSP1 and normal VEGF-A levels could represent a selective target for antiangiogenic therapies.

BACKGROUND

Left ventricular heart failure (LVHF) remains progressive and fatal and is a formidable health problem because ever-larger numbers of people are diagnosed with this disease. Therapeutics, while relieving symptoms and extending life in some cases, cannot resolve this process and transplant remains the option of last resort for many. Our team has described a widely expressed cell surface receptor (CD47) that is activated by its high-affinity secreted ligand, thrombospondin 1 (TSP1), in acute injury and chronic disease; however, a role for activated CD47 in LVHF has not previously been proposed.

RESULTS

In experimental LVHF TSP1-CD47 signaling is increased concurrent with up-regulation of cardiac histone deacetylase 3 (HDAC3). Mice mutated to lack CD47 displayed protection from transverse aortic constriction (TAC)-driven LVHF with enhanced cardiac function, decreased cellular hypertrophy and fibrosis, decreased maladaptive autophagy, and decreased expression of HDAC3. In cell culture, treatment of cardiac myocyte CD47 with a TSP1-derived peptide, which binds and activates CD47, increased HDAC3 expression and myocyte hypertrophy in a Ca(2+)/calmodulin protein kinase II (CaMKII)-dependent manner. Conversely, antibody blocking of CD47 activation, or pharmacologic inhibition of CaMKII, suppressed HDAC3 expression, decreased myocyte hypertrophy, and mitigated established LVHF. Downstream gene suppression of HDAC3 mimicked the protective effects of CD47 blockade and decreased hypertrophy in myocytes and mitigated LVHF in animals.

CONCLUSIONS

These data identify a proximate role for the TSP1-CD47 axis in promoting LVHF by CaKMII-mediated up-regulation of HDAC3 and suggest novel therapeutic opportunities.

Although human small cell lung carcinoma (SCLC) cell lines are typically anchorage-independent and do not attach on most extracellular matrix proteins, OH-1, and several other SCLC cell lines attached on substrates coated with thrombospondin-1 (TSP1). SCLC cells grew long-term as adherent cells on a TSP1-coated substrate. Adhesion of SCLC cells on TSP1 was inhibited by heparin, function-blocking antibodies recognizing alpha3 or beta1 integrin subunits, and by soluble alpha3beta1 integrin ligands. SCLC cells extended neurite-like processes on a TSP1 substrate, which was also mediated by alpha3beta1 integrin. Process formation on a TSP1 substrate was specifically stimulated by epidermal growth factor and somatostatin. Adhesion on TSP1 weakly inhibited SCLC cell proliferation, but this inhibition was strongly enhanced in the presence of epidermal growth factor. TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 also inhibited proliferation when added in solution. High-affinity binding of 125I-labeled TSP1 to OH-1 cells was heparin-dependent and may be mediated by sulfated glycolipids, which are the major sulfated glycoconjugates synthesized by these cells. Synthesis or secretion of TSP1 by SCLC cells could not be detected. On the basis of these results, the alpha3beta1 integrin and sulfated glycolipids cooperate to mediate adhesion of SCLC cells on TSP1. Interaction with TSP1 through this integrin inhibits growth and induces neurotypic differentiation, which suggests that this response to TSP1 may be exploited to inhibit the progression of SCLC.

Previously, we demonstrated that macrophages from thrombospondin 1 (TSP1)-deficient mice have a reduced inflammatory phenotype, suggesting that TSP1 plays a role in macrophage activation. In this study, we determined how TSP1 regulates macrophage function. We found that recombinant or purified platelet human TSP1 treatment stimulated tumor-necrosis factor (TNF)-α expression in bone marrow-derived macrophages in a time- and dose-dependent manner. Toll-like receptor 4 (TLR4) expression (at the mRNA and protein levels) and nuclear factor-kappaB (NF-κB) activity were also stimulated by TSP1 treatment. The TSP1-mediated increase in TNF-α production was abolished in TLR4-deficient macrophages, suggesting that TSP1 activates macrophages through a TLR4-dependent pathway. TSP1 also stimulated TLR4 activation in macrophages in vivo. Furthermore, TSP1-mediated macrophage activation was attenuated by using a peptide or an antibody to block the association between TSP1 and CD36. Taken together, these data suggest that the stimulation of the macrophage TLR4 pathway by TSP1 is partially mediated by the interaction of TSP1 with its receptor, CD36.

Cell-cell and cell-matrix interactions play important regulatory roles in lymphocyte homeostasis. Thrombospondin-1 (TSP1) is a matricellular protein that differentially promotes the adhesion of resting and activated T cells. In this work, we show that adhesion of Jurkat T cells on substrates coated with TSP1 or TSP1-derived peptides is mediated by beta 1 integrins, CD47, and heparan sulfate proteoglycans. Interactions with TSP1 or TSP1 peptides stimulated CD3-induced Ras activation and tyrosine phosphorylation of several T cell proteins. The signals from TSP1 and its derived peptides differentially synergized with activation of the TCR to induce phosphorylation of linker for activation of T cells (LAT) and extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38 kinases. The phosphorylation of ERK in the presence of full-length TSP1 was transient and dependent on a beta 1 integrin receptor. Interestingly, peptides derived from the type 1 repeats of TSP1 and a CD47-binding peptide from the carboxyl-terminal domain of TSP1 also stimulated mitogen-activated protein (MAP) kinase phosphorylation. Moreover, the TSP1 heparin-binding peptide synergized with Ab-ligated TCR to transduce signals to the nucleus, detected by activation of AP-1- and Elk-dependent transcription. This TSP1 peptide-dependent activation of AP-1 was inhibited by both heparin and the MAP/ERK kinase inhibitor PD98059, providing a functional link between adhesion molecule interaction and nuclear transactivation events via the MAP kinase pathways. These findings have implications for the role of extracellular TSP1 and TSP1 fragments in the regulation of T cell function during hemostasis, wound repair, and other inflammatory responses.

Cryptosporidium parvum is recognized as an enteropathogen of great worldwide medical and veterinary importance, yet understanding of its pathogenesis has been hampered in part by limited knowledge of the invasion machinery of this parasite. Recently, genes containing thrombospondin type 1 (<em>TSP1</em>) domains have been identified in several genera of apicomplexans, including thrombospondin-related adhesive proteins (TRAPs) that have been implicated as key molecules for parasite motility and adhesion onto host cell surfaces. Previously, a large-scale random survey of the C. parvum genome conducted in our laboratory revealed the presence of multiple genomic DNA sequences with a high degree of similarity to known apicomplexan TRAP genes. In the present study, TBLASTN screening of available C. parvum genomic sequences by using <em>TSP1</em> domains as queries identified a total of 12 genes possessing <em>TSP1</em>-like domains. All genes have putative signal peptide sequences, one or more <em>TSP1</em>-like domains, plus additional extracellular protein modules such as Kringle, epidermal growth factor, and Apple domains. Two genes, putative paralogs CpTSP8 and CpTSP9, contain predicted introns near their amino termini, which were verified by comparing PCR products from cDNA versus genomic DNA templates. Reverse transcription-PCR analysis of transcript levels reveals that C. parvum TSP genes were developmentally regulated with distinct patterns of expression during in vitro infection. TRAPC1, CpTSP3, and Cp<em>TSP1</em>1 were expressed at high levels during both early and late stages of infection, whereas CpTSP2, CpTSP5, CpTSP6, CpTSP8, and CpTSP9 were maximally expressed during the late stages of infection. Only CpTSP4 was highly expressed solely at an early stage of infection.

CD47 signaling in endothelial cells has been shown to suppress angiogenesis, but little is known about the link between CD47 and endothelial senescence. Herein, we demonstrate that the thrombospondin-1 (TSP1)-CD47 signaling pathway is a major mechanism for driving endothelial cell senescence. CD47 deficiency in endothelial cells significantly improved their angiogenic function and attenuated their replicative senescence. Lack of CD47 also suppresses activation of cell cycle inhibitors and upregulates the expression of cell cycle promoters, leading to increased cell cycle progression. Furthermore, TSP1 significantly accelerates replicative senescence and associated cell cycle arrest in a CD47-dependent manner. These findings demonstrate that TSP1-CD47 signaling is an important mechanism driving endothelial cell senescence. Thus, TSP1 and CD47 provide attractive molecular targets for treatment of aging-associated cardiovascular dysfunction and diseases involving endothelial dysregulation.

BACKGROUND

Histological examination of abdominal aortic aneurysm (AAA) tissues demonstrates extracellular matrix destruction and infiltration of inflammatory cells. Previous work with mouse models of AAA has shown that anti-inflammatory strategies can effectively attenuate aneurysm formation. Thrombospondin-1 is a matricellular protein involved in the maintenance of vascular structure and homeostasis through the regulation of biological functions, such as cell proliferation, apoptosis, and adhesion. Expression levels of thrombospondin-1 correlate with vascular disease conditions.

OBJECTIVE

To use thrombospondin-1-deficient (Thbs1(-/-)) mice to test the hypothesis that thrombospondin-1 contributes to pathogenesis of AAAs.

RESULTS

Mouse experimental AAA was induced through perivascular treatment with calcium phosphate, intraluminal perfusion with porcine elastase, or systemic administration of angiotensin II. Induction of AAA increased thrombospondin-1 expression in aortas of C57BL/6 or apoE-/- mice. Compared with Thbs1(+/+) mice, Thbs1(-/-) mice developed significantly smaller aortic expansion when subjected to AAA inductions, which was associated with diminished infiltration of macrophages. Thbs1(-/-) monocytic cells had reduced adhesion and migratory capacity in vitro compared with wild-type counterparts. Adoptive transfer of Thbs1(+/+) monocytic cells or bone marrow reconstitution rescued aneurysm development in Thbs1(-/-) mice.

CONCLUSIONS

Thrombospondin-1 expression plays a significant role in regulation of migration and adhesion of mononuclear cells, contributing to vascular inflammation during AAA development.

Thrombospondin-1 (TSP1) is an endogenous inhibitor of angiogenesis, which limits blood vessel density in normal tissues and curtails tumor growth. Previous studies of the molecular and cellular effects of TSP1 in angiogenesis have been contradictory. Here, we show that retinal endothelial cells (REC) prepared from TSP1-deficient (TSP1-/-) mice are more proliferative and migratory compared to the wild type REC. We observed up-regulation of the cell cycle regulators, including cyclin A, D1, and Cdk2, as well as the enhanced sequential activities of Src, PI3-kinase, Akt/PKB, Rac1/Cdc42 GTPases, and p38 MAP kinase in TSP1-/- REC. The increased levels of fibronectin and active Akt/PKB were also observed in retinal vasculature of TSP1-/- mice in vivo. Inhibition of Src/PI3-kinase/P38 MAP kinase activities in TSP1-/- REC resulted in decreased migration. Furthermore, TSP1-/- REC showed decreased intracellular levels of active Fyn and JNK2 without affecting caspase-3 activity. Thus, our results demonstrate that in the absence of TSP1, the proangiogenic signaling is enhanced, possibly through up-regulation of fibronectin expression. The enhanced signaling further promotes EC proliferation, migration, and survival. These novel observations support the TSP1's role as an endogenous inhibitor of angiogenesis whose endothelium expression promotes a quiescent, differentiated phenotype.

OBJECTIVE

To assess the impact of thrombospondin 1(TSP1) deficiency on choroidal neovascularization (CNV)and to determine whether administration of a TSP1 antiangiogenic mimetic peptide attenuates CNV.

METHODS

The impact of TSP1 deficiency on laser induced CNV was assessed using wild-type (TSP1 +/+) and TSP1-deficient (TSP1 −/−) mice. Three laser burns were placed in each eye of TSP1 +/+ and TSP1 −/− mice to induce CNV. Intravitreal injection of the TSP1 mimetic peptide was performed on days 1 and 7 postlaser in the mice.For quantitative measurements of neovascularization, intercellular adhesion molecule 2 staining was performed at 14 days postlaser of the choroidal-sclera flat mounts. The recruitment of macrophages to the sites of damage was investigated by immunohistochemistry. The CNV area was measured by intercellular adhesion molecule 2 staining and use of ImageJ software.

RESULTS

The TSP1 −/− mice exhibited significantly larger areas of neovascularization on choroidal flat mounts compared with TSP1 +/ mice. This was consistent with enhanced recruitment of macrophages in TSP1 −/− mice compared with TSP1 +/+ mice 3 days postlaser. The development of CNV was significantly attenuated in mice receiving the TSP1 antiangiogenic mimetic peptide compared with those receiving vehicle alone.

CONCLUSIONS

Deficiency of TSP1 contributes to enhanced choroidal neovascularization. This is consistent with the anti-inflammatory and antiangiogenic activity of TSP1. The TSP1 antiangiogenic peptide was effective in attenuation of CNV.

CONCLUSIONS

Intravitreal injection of TSP1 antiangiogenic mimetic peptides may provide alternative treatment for CNV.

Targeted therapies aimed at tumor vasculature are utilized in combination with chemotherapy to improve drug delivery and efficacy after tumor vascular normalization. Tumor vessels are highly disorganized with disrupted blood flow impeding drug delivery to cancer cells. Although pharmacologic anti-angiogenic therapy can remodel and normalize tumor vessels, there is a limited window of efficacy and these drugs are associated with severe side effects necessitating alternatives for vascular normalization. Recently, moderate aerobic exercise has been shown to induce vascular normalization in mouse models. Here, we provide a mechanistic explanation for the tumor vascular normalization induced by exercise. Shear stress, the mechanical stimuli exerted on endothelial cells by blood flow, modulates vascular integrity. Increasing vascular shear stress through aerobic exercise can alter and remodel blood vessels in normal tissues. Our data in mouse models indicate that activation of calcineurin-NFAT-TSP1 signaling in endothelial cells plays a critical role in exercise-induced shear stress mediated tumor vessel remodeling. We show that moderate aerobic exercise with chemotherapy caused a significantly greater decrease in tumor growth than chemotherapy alone through improved chemotherapy delivery after tumor vascular normalization. Our work suggests that the vascular normalizing effects of aerobic exercise can be an effective chemotherapy adjuvant.

Mesenchymal stem cells (MSCs) are powerful immunomodulators that regulate the diverse functions of immune cells involved in allogeneic reactions, such as T cells and natural killer cells (NK), through cell-cell contact or secreted factors. Exosomes secreted by MSCs may be involved in their regulatory functions, providing new therapeutic tools. Here, we showed that fetal liver (FL) MSC-derived exosomes inhibit proliferation, activation, and cytotoxicity of NK cells. Exosomes bearing LAP, TGFβ, and TSP1, a regulatory molecule for TGFβ, induced downstream TGFβ/Smad2/3 signaling in NK cells. The inhibition of TGFβ using a neutralizing anti-TGFβ antibody, restored NK proliferation, differentiation, and cytotoxicity, demonstrating that FL-MSC-derived exosomes exert their inhibition on NK cell function via TGFβ. These results suggest that FL-MSC-derived exosomes regulate NK cell functions through exosome associated TGFβ.

The expression vectors of the protozoan parasite Trypanosoma cruzi pRIBOTEX and pTREX harbor a ribosomal promoter that improves gene expression and clone selection. Interestingly, the solely presence of this 810 bp long sequence leads to the integration of these vectors into the ribosomal locus, even though circular plasmids are poorly recombinogenic. Initially, it was suggested that a 174 bp long ribosomal-specific repeat element present in the ribosomal promoter region could be responsible for the genetic exchange. On the contrary, we demonstrate that recombination of pTREX occurs within a 86 bp long region located 120 bp downstream the transcription start point (tsp1) of the ribosomal promoter, and it does not depend on the presence of the ribosomal repeat. We also determined that a 291 bp segment encompassing the tsp1 and the 86 bp long recombination region contains all necessary signals to drive transcription and complete recombination into the rRNA locus. Finally, we demonstrate that the integration of pTREX derived plasmids into the nuclear genome occurs within the first 5 h post-transfection, and that non-integrated copies are rapidly degraded.

BACKGROUND

Thrombotic thrombocytopenic purpura (TTP) is most commonly associated with deficiency or inhibition of von Willebrand factor-cleaving protease (ADAMTS-13) activity. ADAMTS-13 mutations and polymorphisms have been reported in childhood congenital TTP, but their significance in adult onset TTP remains unclear.

OBJECTIVE

We sought to identify common ADAMTS-13 mutations in adults with late onset TTP and to investigate whether they may predispose acute clinical episodes of the disorder in adulthood.

UNASSIGNED

We detected a missense mutation (C3178T) in exon 24 of ADAMTS-13 in 6/53 (11.3%) adult onset TTP patients, but no normal controls (n = 100). Three of the patients had pregnancy-associated TTP; three had chronic relapsing acute idiopathic TTP. C3178T encodes an arginine to tryptophan (R1060W) substitution in the TSP1-7 domain of ADAMTS-13. In vitro expression of mutant and wild-type ADAMTS-13 demonstrated that R1060W caused severe intracellular retention of ADAMTS-13 (<5% secretion) without affecting its metalloprotease activity. One homozygous and five heterozygous patients were identified. No other causative mutations were discovered, yet all six patients had ADAMTS-13 activity levels <5% at presentation (normal: 66-126%). Antibodies/inhibitors to ADAMTS-13 were detected in three/five heterozygous patients, and all six patients had subnormal antigen levels. Six asymptomatic first-degree relatives, including those of two probands with antibodies, were also heterozygous for C3178T; all but one had subnormal ADAMTS-13 activity.

CONCLUSIONS

The high prevalence of R1060W ADAMTS-13 in adult onset TTP, together with its absence in childhood congenital TTP cases reported elsewhere, suggests it may be a factor in the development of late onset TTP.

Thrombospondin 1 (TSP1) is a homotrimeric glycoprotein composed of 150-kDa subunits connected by disulfide bridges. The procollagen module of thrombospondin 1 has been implicated in antiangiogenic activity. Procollagen modules are found in a number of extracellular proteins and are identifiable by 10 cysteines with characteristic spacing. We expressed and studied the procollagen module (C) of human TSP1, both by itself and in the context of the adjoining oligomerization sequence (o) and N-terminal module (N). The coding sequences were introduced into baculoviruses along with an N-terminal signal sequence and C-terminal polyhistidine tag. Proteins were purified from conditioned medium of infected insect cells by nickel-chelate chromatography. NoC is a disulfide bonded trimer and cleaves readily at a site of preferential proteolysis to yield monomeric N and trimeric oC. These are known properties of full-length TSP1. Mass spectroscopy indicated that C is N-glycosylated, and all 10 cysteine residues of C are in disulfides. By equilibrium ultracentrifugation, C is a monomer in physiological salt solution. Circular dichroism, intrinsic fluorescence, and differential scanning calorimetry experiments suggest that the stability of C is determined by the disulfides. The two tryptophans of C are in a polar, exposed environment as assessed by iodide fluorescence quenching and solvent perturbation. The oC far UV circular dichroism spectrum could be modeled as the sum of C and a coiled-coil oligomerization domain. The results indicate that the recombinant C folds autonomously into its native structure, and trimerization of the modules in TSP1 does not perturb their structures.

Gabapentin (GBP) is an anticonvulsant that acts at the α2δ-1 submit of the L-type calcium channel. It is recently reported that GBP is a potent inhibitor of thrombospondin (TSP)-induced excitatory synapse formation in vitro and in vivo. Here we studied effects of chronic GBP administration on epileptogenesis in the partial cortical isolation ("undercut") model of posttraumatic epilepsy, in which abnormal axonal sprouting and aberrant synaptogenesis contribute to occurrence of epileptiform discharges. Results showed that 1) the incidence of evoked epileptiform discharges in undercut cortical slices studied 1 day or ~2 weeks after the last GBP dose, was significantly reduced by GBP treatments, beginning on the day of injury; 2) the expression of GFAP and TSP1 protein, as well as the number of FJC stained cells was decreased in GBP treated undercut animals; 3) in vivo GBP treatment of rats with undercuts for 3 or 7 days decreased the density of vGlut1-PSD95 close appositions (presumed synapses) in comparison to saline treated controls with similar lesions;4) the electrophysiological data are compatible with the above anatomical changes, showing decreases in mEPSC and sEPSC frequency in the GBP treated animals. These results indicate that chronic administration of GBP after cortical injury is antiepileptogenic in the undercut model of post-traumatic epilepsy, perhaps by both neuroprotective actions and decreases in excitatory synapse formation. The findings may suggest the potential use of GBP as an antiepileptogenic agent following traumatic brain injury.

BACKGROUND

We have previously evaluated the vaccine efficacies of seven tetraspanins of Echinococcus multilocularis (Em-TSP1-7) against alveolar echinococcosis (AE) by subcutaneous (s.c.) administration with Freund's adjuvant. Over 85% of liver cyst lesion number reductions (CLNR) were achieved by recombinant Em-TSP1 (rEm-TSP1) and -TSP3 (rEm-TSP3). However, to develop an efficient and safe human vaccine, the efficacy of TSP mucosal vaccines must be thoroughly evaluated.

RESULTS

rEm-TSP1 and -TSP3 along with nontoxic CpG ODN (CpG oligodeoxynucleotides) adjuvant were intranasally (i.n.) immunized to BALB/c mice and their vaccine efficacies were evaluated by counting liver CLNR (experiment I). 37.1% (p < 0.05) and 62.1% (p < 0.001) of CLNR were achieved by these two proteins, respectively. To study the protection-associated immune responses induced by rEm-TSP3 via different immunization routes (i.n. administration with CpG or s.c. immunization with Freund's adjuvant), the systemic and mucosal antibody responses were detected by ELISA (experiment II). S.c. and i.n. administration of rEm-TSP3 achieved 81.9% (p < 0.001) and 62.8% (p < 0.01) CLNR in the liver, respectively. Both the immunization routes evoked strong serum IgG, IgG1 and IgG2α responses; i.n. immunization induced significantly higher IgA responses in nasal cavity and intestine compared with s.c. immunization (p < 0.001). Both immunization routes induced extremely strong liver IgA antibody responses (p < 0.001). The Th1 and Th2 cell responses were assessed by examining the IgG1/IgG2α ratio at two and three weeks post-immunization. S.c. immunization resulted in a reduction in the IgG1/IgG2α ratio (Th1 tendency), whereas i.n. immunization caused a shift from Th1 to Th2. Moreover, immunohistochemistry showed that Em-TSP1 and -TSP3 were extensively located on the surface of E. multilocularis cysts, protoscoleces and adult worms with additional expression of Em-TSP3 in the inner part of protoscoleces and oncospheres.

CONCLUSIONS

Our study indicated that i.n. administration of rEm-TSP3 with CpG is able to induce both systemic and local immune responses and thus provides significant protection against AE.

Opioid receptor signaling via EGF receptor (EGFR) transactivation and ERK/MAPK phosphorylation initiates diverse cellular responses that are cell type-dependent. In astrocytes, multiple μ opioid receptor-mediated mechanisms of ERK activation exist that are temporally distinctive and feature different outcomes. Upon discovering that chronic opiate treatment of rats down-regulates thrombospondin 1 (TSP1) expression in the nucleus accumbens and cortex, we investigated the mechanism of action of this modulation in astrocytes. TSP1 is synthesized in astrocytes and is released into the extracellular matrix where it is known to play a role in synapse formation and neurite outgrowth. Acute morphine (hours) reduced TSP1 levels in astrocytes. Chronic (days) opioids repressed TSP1 gene expression and reduced its protein levels by μ opioid receptor and ERK-dependent mechanisms in astrocytes. Morphine also depleted TSP1 levels stimulated by TGFβ1 and abolished ERK activation induced by this factor. Chronic morphine treatment of astrocyte-neuron co-cultures reduced neurite outgrowth and synapse formation. Therefore, inhibitory actions of morphine were detected after both acute and chronic treatments. An acute mechanism of morphine signaling to ERK that entails depletion of TSP1 levels was suggested by inhibition of morphine activation of ERK by a function-blocking TSP1 antibody. This raises the novel possibility that acute morphine uses TSP1 as a source of EGF-like ligands to activate EGFR. Chronic morphine inhibition of TSP1 is reminiscent of the negative effect of μ opioids on EGFR-induced astrocyte proliferation via a phospho-ERK feedback inhibition mechanism. Both of these variations of classical EGFR transactivation may enable opiates to diminish neurite outgrowth and synapse formation.

Obesity is prevalent worldwide and is a major risk factor for many diseases including renal complications. Thrombospondin 1 (TSP1), a multifunctional extracellular matrix protein, plays an important role in diabetic kidney diseases. However, whether TSP1 plays a role in obesity-related kidney disease is unknown. In the present studies, the role of TSP1 in obesity-induced renal dysfunction was determined by using a diet-induced obese mouse model. The results demonstrated that TSP1 was significantly upregulated in the kidney from obese mice. The increased TSP1 was localized in the glomerular mesangium as well as in the tubular system from obese wild-type mice. Obese wild-type mice developed renal hypertrophy and albuminuria, which was associated with increased kidney macrophage infiltration, augmented kidney inflammation, and activated transforming growth factor (TGF)-β signaling and renal fibrosis. In contrast, obese TSP1-deficient mice did not develop these kidney damages. Furthermore, in vitro studies demonstrated that leptin treatment stimulated the expression of TSP1, TGF-β1, fibronectin, and collagen type IV in mesangial cells isolated from wild-type mice. These leptin-stimulated effects were abolished in TSP1-deficient mesangial cells. Taken together, these data suggest that TSP1 is an important mediator for obesity- or hyperleptinemia-induced kidney dysfunction.