Hyperglycemia is a crucial factor in the development of diabetic nephropathy. We previously showed that high glucose upregulates thrombospondin 1 (TSP1)-dependent transforming growth factor (TGF)-beta activation by altering cGMP-dependent protein kinase (PKG) activity as a result of decreased nitric oxide signaling. In the present study, we showed that high glucose concentrations significantly reduced endogenous PKG activity. To further examine the mechanisms by which PKG regulates TSP1 expression and TSP1-dependent TGF-beta activation, we generated stably transfected rat mesangial cells (RMCs) with inducible expression tetracycline-induced gene expression of the catalytic domain of PKG. After tetracycline induction, the catalytic domain of PKG is expressed as a cGMP-independent active kinase. Expression of the catalytic domain prevented high glucose-mediated increases in transcription of the TSP1 gene with no alteration in TSP1 mRNA stability. Glucose stimulation of TSP1 protein expression and TGF-beta bioactivity were also downregulated. TGF-beta-dependent fibronectin and type IV collagen expression under high glucose conditions were significantly reduced upon catalytic domain expression in transfected RMCs. These results show that constitutively active PKG inhibits the fibrogenic potential of high glucose through repression of TSP1-dependent TGF-beta bioactivity, suggesting that gene transfer of the catalytic domain of PKG might provide a new strategy for treatment of diabetic renal fibrosis.

BACKGROUND

ADAMTS-7, a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, was recently identified to be significantly associated genomewide with coronary artery disease. However, the mechanisms that link ADAMTS-7 and coronary artery disease risk remain elusive. We have previously demonstrated that ADAMTS-7 promotes vascular smooth muscle cell migration and postinjury neointima formation via degradation of a matrix protein cartilage oligomeric matrix protein. Because delayed endothelium repair renders neointima and atherosclerosis plaque formation after vessel injury, we examined whether ADAMTS-7 also inhibits re-endothelialization.

RESULTS

Wire injury of the carotid artery and Evans blue staining were performed in Adamts7(-/-) and wild-type mice. Adamts-7 deficiency greatly promoted re-endothelialization at 3, 5, and 7 days after injury. Consequently, Adamts-7 deficiency substantially ameliorated neointima formation in mice at days 14 and 28 after injury in comparison with the wild type. In vitro studies further indicated that ADAMTS-7 inhibited both endothelial cell proliferation and migration. Surprisingly, cartilage oligomeric matrix protein deficiency did not affect endothelial cell proliferation/migration and re-endothelialization in mice. In a further examination of other potential vascular substrates of ADAMTS-7, a label-free liquid chromatography-tandem mass spectrometry secretome analysis revealed thrombospondin-1 as a potential ADAMTS-7 target. The subsequent studies showed that ADAMTS-7 was directly associated with thrombospondin-1 by its C terminus and degraded thrombospondin-1 in vivo and in vitro. The inhibitory effect of ADAMTS-7 on postinjury endothelium recovery was circumvented in Tsp1(-/-) mice.

CONCLUSIONS

Our study revealed a novel mechanism by which ADAMTS-7 affects neointima formation. Thus, ADAMTS-7 is a promising treatment target for postinjury vascular intima hyperplasia.

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NFkappaB, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NFkappaB suppression, since it was restricted to the cells lacking nuclear (active) NFkappaB. Thus we for the first time identified combined decrease of NFkappaB and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. (c) 2007 Wiley-Liss, Inc.

Cnidarians represent the first animal phylum with an organized nervous system and a complex active behavior. The hydra nervous system is formed of sensory-motoneurons, ganglia neurons and mechanoreceptor cells named nematocytes, which all differentiate from a common stem cell. The neurons are organized as a nerve net and a subset of neurons participate in a more complex structure, the nerve ring that was identified in most cnidarian species at the base of the tentacles. In order to better understand the genetic control of this neuronal network, we analysed the expression of evolutionarily conserved regulatory genes in the hydra nervous system. The Prd-class homeogene prdl-b and the nuclear orphan receptor hyCOUP-TF are expressed at strong levels in proliferating nematoblasts, a lineage where they were found repressed during patterning and morphogenesis, and at low levels in distinct subsets of neurons. Interestingly, Prd-class homeobox and COUP-TF genes are also expressed during neurogenesis in bilaterians, suggesting that mechanoreceptor and neuronal cells derive from a common ancestral cell. Moreover, the Prd-class homeobox gene prdl-a, the Antp-class homeobox gene msh, and the thrombospondin-related gene TSP1, which are expressed in distinct subset of neurons in the adult polyp, are also expressed during early budding and/or head regeneration. These data strengthen the fact that two distinct regulations, one for neurogenesis and another for patterning, already apply to these regulatory genes, a feature also identified in bilaterian related genes.

BACKGROUND

Remodeling of the extracellular matrix plays an important role during the pathogenesis of atherosclerosis and restenosis. The matrix glycoprotein thrombospondin-1 (TSP1) inhibits endothelial cell proliferation and migration in vitro. In contrast, TSP1 facilitates the growth and migration of cultured vascular smooth muscle cells. Accordingly, we investigated the hypothesis that administration of anti-TSP1 antibody could facilitate reendothelialization and inhibit neointimal thickening in balloon-injured rat carotid artery.

RESULTS

Sprague-Dawley rats were subjected to left common carotid artery denudation, after which arteries were treated with C6.7 anti-TSP1 or control antibody. Evans blue dye staining 2 weeks after injury disclosed significantly increased reendothelialization in arteries treated with C6.7 antibody compared with the control group, and this effect was associated with increased number of proliferating cell nuclear antigen-positive endothelial cells. In contrast, treatment with C6.7 antibody decreased the number of proliferating cell nuclear antigen-positive vascular smooth muscle cells in the injured arterial wall. Neointimal thickening was correspondingly attenuated to a statistically significant degree in arteries receiving C6.7 antibody versus the control group at both the 2-week and 4-week time points.

CONCLUSIONS

Intra-arterial delivery of antibody against TSP1 facilitated reendothelialization and reduced neointimal lesion formation after balloon denudation.

Astrocytes contribute to many neuronal functions, including synaptogenesis, but their role in the development of synaptic plasticity remains unclear. Presynaptic muting of hippocampal glutamatergic terminals defends against excitotoxicity. Here we studied the role of astrocytes in the development of presynaptic muting at glutamatergic synapses in rat hippocampal neurons. We found that astrocytes were critical for the development of depolarization-dependent and G(i/o)-dependent presynaptic muting. The ability of cAMP analogues to modulate presynaptic function was also impaired by astrocyte deficiency. Although astrocyte deprivation resulted in postsynaptic glutamate receptor deficits, this effect appeared independent of astrocytes' role in presynaptic muting. Muting was restored with chronic, but not acute, treatment with astrocyte-conditioned medium, indicating that a soluble factor is permissive for muting. Astrocyte-derived thrombospondins (TSPs) are likely responsible because TSP1 mimicked the effect of conditioned medium, and gabapentin, a high-affinity antagonist of TSP binding to the α2δ-1 calcium channel subunit, mimicked astrocyte deprivation. We found evidence that protein kinase A activity is abnormal in astrocyte-deprived neurons but restored by TSP1, so protein kinase A dysfunction may provide a mechanism by which muting is disrupted during astrocyte deficiency. In summary our results suggest an important role for astrocyte-derived TSPs, acting through α2δ-1, in maturation of a potentially important form of presynaptic plasticity.

Microvascular development is often perceived to result from a balance of positive and negative factors that impact signaling for proliferation and survival. The survival signaling that results from hypoxia-induced VEGF-A has been well established, but the factors that antagonize this signaling have been poorly studied. As endogenous inhibitors of angiogenesis, thrombospondins (TSPs) are likely candidates to affect survival signaling. Here we report that TSP1 antagonized microvascular survival to retinal hyperoxia, and Akt signaling in both the retina and in cultured endothelial cells. TSP1 expression is correlated with the association of the CD36 receptor with Src versus Fyn. In the presence of TSP1, CD36 is coprecipitated with Fyn as previously shown by others. However, in the absence of TSP1, there is a preferential association with Src. We now demonstrate that these Src family kinases play an important role in modulating microvascular survival in response to TSP1 by crossing tsp1(-/-) mice to the src(-/-) and fyn(-/-) mice and testing the survival of retinal blood vessels in hyperoxia. We find that tsp1(-/-), fyn(-/-), and double-mutant tsp1(-/-)/fyn(-/-) mice have a similar enhancement of capillary survival in oxygen, whereas in a tsp(-/-) background, the loss of only one allele of src restores the balance in survival and apoptosis to that of wild-type mice. Taken together, we hypothesize that TSP1 antagonizes VEGF-driven Akt survival signaling in part through the recruitment of Fyn to membrane domains containing CD36, but when TSP1 is absent, an opposing Src recruitment contributes to VEGF-driven Akt phosphorylation and capillary survival.

Renal cell carcinoma (RCC), the most common type of kidney cancer, currently has no biomarker of clinical utility. The present study utilized a mass spectrometry-based proteomics workflow for identifying differentially abundant proteins in RCC by harvesting shed and secreted proteins from the tumor microenvironment through sampling tissue interstitial fluid (TIF) from radical nephrectomies. Matched tumor and adjacent normal kidney (ANK) tissues were collected from 10 patients diagnosed with clear cell RCC. One-hundred thirty-eight proteins were identified with statistically significant differential abundances derived by spectral counting in tumor TIF when compared to ANK TIF. Among those proteins with elevated abundance in tumor TIF, nicotinamide n-methyltransferase (NNMT) and enolase 2 (ENO2) were verified by Western blot and selected reaction monitoring (SRM). The presence of ENO2 and thrombospondin-1 (TSP1) were verified as present and at elevated abundance in RCC patient serum samples as compared to a pooled standard control by enzyme-linked immunosorbent assay (ELISA), recapitulating the relative abundance increase in RCC as compared with ANK TIF.

BACKGROUND

The cysteine-rich/spacer domains of ADAMTS13 contain a major binding site for antibodies in patients with acquired thrombotic thrombocytopenic purpura (TTP).

OBJECTIVE

To study the heterogeneity of the antibody response towards these domains an immunoglobulin V-gene phage-display library was constructed to isolate monoclonal anti-ADAMTS13 antibodies from the immunoglobulin repertoire of a patient with acquired TTP.

METHODS

Combined variable heavy chain (VH) and variable light chain (VL) segments, expressed as single-chain Fv fragments (scFv), were selected for binding to an ADAMTS13 fragment consisting of the disintegrin/thrombospondin type-1 repeat 1 (TSP1)/cysteine-rich/spacer domains.

RESULTS

Seven different scFv antibody clones were identified that were assigned to four groups based on their homology to VH germline gene segments. Epitope-mapping revealed that scFv I-9 (VH1-69), I-26 (VH1-02), and I-41 (VH3-09) bind to an overlapping binding site in the ADAMTS13 spacer domain, whereas scFv I-16 (VH3-07) binds to the disintegrin/TSP1 domains. The affinity of scFv for the disintegrin/TSP1/cysteine-rich/spacer domain was determined by surface plasmon resonance analysis and the dissociation constants ranged from 3 to 254 nM. The scFv partially inhibited ADAMTS13 activity. However, full-length IgG prepared from the variable domains of scFv I-9 inhibited ADAMTS13 activity more profoundly. Plasma of six patients with acquired TTP competed for binding of scFv I-9 to ADAMTS13.

CONCLUSIONS

Our data indicate that multiple B-cell clones producing antibodies directed against the spacer domain are present in the patient analyzed in this study. Our findings also suggest that antibodies with a similar epitope specificity as scFv I-9 are present in plasma of other patients with acquired TTP.

Colorectal cancer arises from the progressive accumulation of mutations and epigenetic alterations in colon epithelial cells. Such alterations often deregulate signaling pathways that affect the formation of colon cancer, such as the Wnt, RAS-MAPK and TGF-beta pathways. The tumor promoting effects of mutations in genes, such as APC, have been demonstrated in cancer cell lines and in mouse models of intestinal cancer; however, the biological effects of most epigenetic events identified in colorectal cancer remain unknown. Consequently, we assessed whether the aberrant methylation of TSP1, the gene for thrombospondin 1, a regulator of TGF-beta ligand activation, is an epigenetic mechanism for inhibiting the TGF-beta signaling pathway. We found methylated TSP1 occurs in colon cancer cell lines (33%), colon adenomas (14%) and colon adenocarcinomas (21%). In primary colorectal cancers, loss of TSP1 expression correlated with impaired TGF-beta signaling as indicated by decreased Smad2 phosphorylation and nuclear localization. Furthermore, methylation-induced silencing of TSP1 expression reduced the concentration of secreted active TGF-beta1 and attenuated TGF-beta signaling. Reversal of TSP1 methylation resulted in increased TSP1 mediated activation of the latent LAP:TGF-beta complex and subsequent TGF-beta receptor activation. Our results demonstrate that the aberrant methylation of TSP1 has biological consequences and provide evidence that the aberrant methylation of TSP1 is a novel epigenetic mechanism for suppressing TGF-beta signaling in colorectal cancer.

Thrombospondin 1 (TSP1) and thrombospondin 2 (TSP2) are members of the thrombospondin family that have a similar structural organization but somewhat different functional activities. Iodinated recombinant mouse TSP2 bound to NIH 3T3 cells and was internalized and degraded through a chloroquine-inhibitable pathway. TSP2 degradation was saturable, specific, and similar to the kinetics of degradation of TSP1. Human platelet TSP1, recombinant mouse TSP1, and recombinant mouse TSP2 cross-competed with one another for degradation by 3T3 cells. Degradation of TSP2 was less sensitive to inhibition by heparin than degradation of TSP1. This is in agreement with differences in heparin-binding affinity of the two TSPs. Degradation of TSP2 was slower in cultures of Chinese hamster ovary (CHO) cells lacking heparan sulfate proteoglycans than in wild type CHO cells or in cultures of 3T3 cells treated with heparitinase than in untreated 3T3 cells. Degradation of TSP2 was inhibited by antibodies against the low density lipoprotein receptor-related protein (LRP) or by the 39-kDa receptor-associated protein, a known antagonist of LRP. This study indicates that TSP2 and TSP1 are metabolized by a common internalization and degradation pathway involving heparan sulfate proteoglycan and LRP. Competition for this pathway is a possible mechanism whereby cells can control the levels and ratio of TSP1 and TSP2 in the extracellular milieu.

CD148 is a receptor-type protein tyrosine phosphatase that is expressed in several cell types, including vascular endothelial cells and duct epithelial cells. Growing evidence demonstrates a prominent role for CD148 in negative regulation of growth factor signals, suppressing cell proliferation and transformation. However, its extracellular ligand(s) remain unknown. To identify the ligand(s) of CD148, we introduced HA-tagged CD148 into cultured endothelial cells and then isolated its interacting extracellular protein(s) by biotin surface labeling and subsequent affinity purifications. The binding proteins were identified by mass spectrometry. Here we report that soluble thrombospondin-1 (TSP1) binds to the extracellular part of CD148 with high affinity and specificity, and its binding increases CD148 catalytic activity, leading to dephosphorylation of the substrate proteins. Consistent with these findings, introduction of CD148 conferred TSP1-mediated inhibition of cell growth to cells which lack CD148 and TSP1 inhibition of growth. Further, we demonstrate that TSP1-mediated inhibition of endothelial cell growth is antagonized by soluble CD148 ectodomain as well as by CD148 gene silencing. These findings provide evidence that CD148 functions as a receptor for TSP1 and mediates its inhibition of cell growth.

The regulator of fumarate and nitrate reduction (FNR) protein of Escherichia coli is an oxygen-responsive transcription regulator that acts mainly to activate the transcription of genes associated with anaerobic energy generation during periods of oxygen starvation. The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae encodes an FNR homologue, HlyX, which can complement the anaerobic respiratory deficiencies of an fnr mutant. However, FNR and HlyX have distinct but overlapping regulons because during anaerobic incubation, hlyX-expressing E. coli K-12 strains produce an otherwise latent haemolysin. The gene encoding the 'latent' haemolysin has been designated hlyE and analysis of the promoter region by DNase I footprinting reveals the presence of an FNR- (HlyX-) binding site. Anaerobic expression of an hlyE::lacZ reporter was 6.5-fold higher in hlyX compared to fnr-expressing cells. Both FNR and HlyX recruited RNA polymerase to the hlyE promoter but formed different ternary complexes. One major transcript (tsp1) initiating at 78.5 bp downstream of the FNR-binding site and four minor transcripts initiating at 73.5 (tsp2), 71.5 (tsp3), 63.5 (tsp4) and 62.5 (tsp5) bp from the FNR site were detected. From the position of the FNR box relative to the transcript starts, hlyE is expressed from a Class I FNR-regulated promoter. Substitution of selected FNR amino acids with the residues found in the equivalent positions in HlyX indicated that Activating Region 1 (AR1) of FNR forms a surface encompassing beta to beta 11 and that the AR1 contact at Class I promoters is different to that at Class II promoters, although the same surface is involved. The FNR variant, FNR-A225T, combined the properties of FNR (good activation from Class II promoters) and HlyX (good activation of Class I promoters) and conferred the haemolytic phenotype.

Metastasis relies on angiogenesis for tumor expansion. Tumor angiogenesis is restrained by a variety of endogenous inhibitors, including thrombospondin 1 (TSP1). The principal antiangiogenic activity of TSP1 resides in a domain containing three TSP1 repeats (3TSR), and TSP1 cleavage is regulated, in part, by the metalloproteinase ADAMTS1. In this study, we examined the role of TSP1 and ADAMTS1 in controlling metastatic disease in the liver and lung. TSP1 overexpression inhibited metastatic growth of colon or renal carcinoma cells in liver but not lung. Metastatic melanoma in liver grew more rapidly in Tsp1-null mice compared with controls, whereas in lung grew similarly in Tsp1-null mice or controls. Recombinant TSP1 was cleaved more efficiently in lysates from liver than lung. ADAMTS1 inhibition by neutralizing antibody, small interfering RNA, or genetic deletion abrogated cleavage activity. To confirm that lack of cleavage of TSP1 ablated its antiangiogenic function in the lung, we generated colon cancer cells stably secreting only the 3TSR domain and found that they inhibited formation of both liver and lung metastases. Collectively, our results indicate that the antiangiogenic activity of TSP1 is differentially regulated by ADAMTS1 in the liver and lung, emphasizing the concept that regulation of angiogenesis is varied in different tissue environments.

Fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis elicit spontaneous proliferation of autologous T cells in an HLA-DR and CD47 costimulation-dependent manner. T cell costimulation through CD47 is attributed to specific interaction with thrombospondin-1 (TSP1), a CD47 ligand displayed on FLS. CD47 binding by FLS has broad biological impact that includes adhesion and the triggering of specific costimulatory signals. TSP1(+) FLS are highly adhesive to T cells and support their aggregation and growth in situ. Long-term cultures of T cells and FLS form heterotypic foci that are amenable to propagation without exogenous growth factors. T cell adhesion and aggregate formation on TSP1(+) FLS substrates are inhibited by CD47-binding peptides. In contrast, FLS from arthroscopy controls lack adhesive or T cell growth-promoting activities. CD47 stimulation transduces a costimulatory signal different from that of CD28, producing a gene expression profile that included induction of ferritin L chain, a component of the inflammatory response. Ferritin L chain augments CD3-induced proliferation of T cells. Collectively, these results demonstrate the active role of FLS in the recruitment, activation, and expansion of T cells in a CD47-dependent manner. Because TSP1 is abundantly expressed in the rheumatoid synovium, CD47-TSP1 interaction is proposed to be a key component of an FLS/T cell regulatory circuit that perpetuates the inflammatory process in the rheumatoid joint.

A number of severe diseases of medical and veterinary importance are caused by parasites of the phylum Apicomplexa. These parasites invade host cells using similar subcellular structures, organelles and molecular species. Proteins containing one or more copies of the type I repeat of human platelet thrombospondin (TSP1), are crucial components of both locomotion and invasion machinery. Members of this family have been identified in Eimeria tenella, E. maxima, Toxoplasma gondii, Cryptosporidium parvum and in all Plasmodium species so far analysed. Here, Andrea Crisanti and colleagues discuss the structure, localization and current understanding of the function of TSP family members in the invasion of target cells by apicomplexan parasites.

ADAMTS13 is the metalloprotease responsible for the proteolytic degradation of von Willebrand factor (VWF). A severe deficiency of this VWF-cleaving protease activity causes thrombotic thrombocytopenic purpura. This protease, comprising 1,427 amino acid residues, is composed of multiple domains, i.e., a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, seven Tsp1 repeats, and two CUB domains. We prepared one polyclonal and seven monoclonal antibodies recognizing distinct epitopes spanning the entire ADAMTS13 molecule. Of these antibodies, two of the monoclonal ones, which recognize the disintegrin-like and cysteine-rich/spacer domains, respectively, abolished the hydrolytic activity of ADAMTS13 toward both a synthetic substrate, FRETS-VWF73, and the natural substrate, VWF. In addition, these antibodies blocked the binding of ADAMTS13 to VWF. These results revealed that the region between the disintegrin-like and cysteine-rich/spacer domains interacts with VWF. Employing these established polyclonal and monoclonal antibodies, we examined the molecular species of ADAMTS13 circulating in the blood by immunoprecipitation followed by Western blot analysis, and estimated the plasma concentration of ADAMTS13 by enzyme-linked immunosorbent assay. These studies indicated that the major fraction of ADAMTS13 in blood plasma consisted of the full-length form. The concentration of ADAMTS13 in normal plasma was approximately 0.5-1 microg/ml.

LIMPII (lysosomal integral membrane protein II) is one of a family of proteins structurally related to the cell surface glycoprotein CD36. We recently defined a single structural domain on CD36 that mediates binding to adhesive glycoprotein thrombospondin-1 (TSP1). The CD36-TSP1 interaction is known to play a role in platelet-tumor and platelet-monocyte adhesion, angiogenesis, and in monocyte uptake of apoptotic cells. To test whether LIMPII also binds TSP1, a LIMPII peptide corresponding to the TSP1 binding domain of CD36 was expressed as a recombinant glutathione S-transferase (GST) fusion protein. In solid phase binding assays, purified 125I-TSP1 bound to immobilized GST/LIMPII in a time-dependent and saturable manner. Inhibition by excess unlabeled TSP1 or EDTA demonstrated specificity. LIMPII.TSP1 complex formation was specifically blocked by soluble LIMPII fusion protein, by monospecific rabbit IgG directed against the LIMPII peptide and by CD36 fusion proteins containing the TSP1 binding domain. Transfection of Bowes melanoma cells with a chimeric LIMPII cDNA that targets expression to the plasma membrane conferred the ability to bind 125I-TSP1 and to adhere to TSP1-coated surfaces. This study defines a TSP1 binding site conserved between LIMPII and CD36 and suggests that cell surface LIMPII may function in some circumstances as an adhesion receptor for TSP1. Computer-assisted homology searches suggest that the TSP1 recognition motif identified from study of CD36 family members may be widely expressed in nature.

A baculovirus system was used to express full-length recombinant mouse thrombospondin 2 (rTSP2) as a disulfide-bonded homotrimer with an NH2 terminus beginning with Asp20.rTSP2, like TSP1, was more sensitive to trypsin digestion if depleted of calcium ion. The trypsin digestion pattern of rTSP2 and TSP1 differed in that trypsin cut between the first and second type 1 modules of rTSP2. For bovine aortic endothelial cells adhering to TSP-coated polystyrene plates, reduction after coating caused both TSPs to be much more adhesive; these adhesions were blocked completely by RGDS peptide or antibody to alpha v beta 3 integrin.rTSP2 and TSP1 also mediated the adhesion of HT-29 human colon adenocarcinoma cells that carry alpha v beta 5 but not alpha v beta 3 integrin. Antibody to alpha v beta 5 did not inhibit adhesion of HT-29 cells to TSP1 or rTSP2. Rather, adhesion of HT-29 cells was decreased by treatment of TSPs with EDTA, abolished by reduction of the TSPs, and, in the case of rTSP2, blocked by heparin. Adhesion of MG63 cells to both TSPs was complex. Treatment with EDTA enhanced the adhesive activity of rTSP2 but decreased the adhesive activity of TSP1. These results show that TSP2 can be processed and secreted when overexpressed using baculovirus, TSP1 and rTSP2 differ in protease susceptibility in the type 1 module region, and TSP1 and rTSP2 mediate cell adhesion by complex and similar but not identical mechanisms.

Diabetic retinopathy is a leading cause of blindness in the United States. Access to new animal models that exhibit retinal vasculopathies with short duration of diabetes, are vital for understanding the underlying mechanisms and examining the efficacy of new treatment modalities. Our previous studies demonstrated decreased expression of Thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis, in eyes from both patients and rodents with diabetes. Here we examined whether TSP1 deficiency could exacerbate diabetic retinal vasculopathies. Akita/+ male mice reproducibly develop diabetes by 4 weeks of age. These mice demonstrated the early non-proliferative stages of diabetic retinopathy, including decreased numbers of pericytes and increased glial cell activation. However, Akita/+ male mice deficient in TSP1 (Akita/+; TSP1-/-) demonstrated more advanced stages of diabetic retinopathy with a 4-fold increase in acellular capillaries and increased fibronectin and GFAP expression. These vascular changes were not attributable to aberrant retinal vascular development in the absence of TSP1, since down-regulation of TSP1 postnatally in the endothelium also resulted in more severe retinopathy. In addition, lack of another endogenous inhibitor of angiogenesis, pigment epithelium derived factor (PEDF), also enhanced diabetic retinopathy in Akita/+ mice. Akita/+; PEDF-/- male mice demonstrated increased numbers of acellular capillaries compared to controls but at a level lower than that observed in Akita/+; TSP1-/- mice. Thus, the exacerbation of diabetic retinopathy in Akita/+; TSP1-/- mice will allow the study of retinal vasculopathies with a shorter duration of diabetes and facilitate future testing of treatment modalities that protect the retinal vasculature and preserve sight.

OBJECTIVE

In vitro data suggest that down-regulation of thrombospondin 1 (TSP1) expression from TXR1 is associated with resistance to taxane-based chemotherapy. The prognostic and predictive value of tumoral expression of both genes was evaluated in patients with lung adenocarcinoma treated with first-line docetaxel and gemcitabine.

METHODS

Tumor samples from 96 patients, with stage IIIB (with pleural effusion) or IV lung adenocarcinomas, were analyzed for TXR1 and TSP1 mRNA levels by quantitative real-time PCR, from microdissected cells derived from patients' primary tumors.

RESULTS

The mRNA levels of the two genes were inversely correlated (Spearman's test = -0.49; P < 0.0001). Patients with low TXR1 mRNA levels experienced a longer median time to tumor progression (TTP; P < 0.0001) and median overall survival (mOS; P = 0.001) when compared with patients with high TXR1 expression. Patients with high TSP1 expression presented longer TTP (P = 0.002) and mOS (P < 0.0001) when compared with patients with low TSP1 expression. Moreover, patients with high TSP1 and low TXR1 expression (n = 36) presented higher prolonged TTP (P = 0.009) and mOS (P < 0.0001) compared with patients with high TXR1 and low TSP1 expression. Multivariate analysis showed that high TXR1/low TSP1 expression was an independent prognostic factor for decreased TTP (hazard ratio, 1.7; 95% confidence interval, 1.1-3.27; P = 0.016) and mOS (hazard ratio, 2.55; 95% confidence interval, 1.57-4.15; P < 0.0001).

CONCLUSIONS

These data confirm the in vitro model of TSP1 and TXR1 effect on taxane resistance in lung adenocarcinomas and merit further evaluation.

Thrombospondins (TSPs) are an evolutionarily ancient family of extracellular calcium-binding glycoproteins. The five mammalian TSPs collectively have important roles in angiogenesis and vascular biology, synaptogenesis, wound repair and connective tissue organisation. Their complex functions relate to the multiple postsecretion fates of TSPs that can involve endocytic uptake, proteolysis or retention within the extracellular matrix (ECM). Surprisingly, the molecular and cellular mechanisms by which TSPs become retained within the ECM are poorly understood. We hypothesised that the highly conserved TSP C-terminal domain mediates ECM retention. We report that ECM incorporation as insoluble punctate deposits is an evolutionarily conserved property of TSPs. ECM retention of TSP1 is mediated by the C-terminal region in trimeric form, and not by C-terminal monomer or trimers of the N-terminal domain or type 1 repeats. Using a novel mRFP-tagged TSP1 C-terminal trimer, we demonstrate that ECM retention involves the RGD site and a novel site in the L-lectin domain with structural similarity to the ligand-binding site of cargo transport proteins. CD47 and beta1 integrins are dispensable for ECM retention, but beta1 integrins enhance activity. These novel data advance concepts of the molecular processes that lead to ECM retention of TSP1.

Angiogenesis plays an important role in growth and proliferation of cancer. Various angiogenic and angiostatic factors regulate angiogenesis. We examined expression of genes encoding various angiostatic factors: thrombospondin 1 (TSP1), thrombospondin 2 (TSP2), brain-specific angiogenesis inhibitor 1 (BAI1) and angiopoietin 2 (AGP2) in 62 colorectal cancers and 40 samples of extraneoplastic colon mucosa. The expression of the angiostatic factors TSP2 and AGP2 were significantly increased in the cancerous mucosa as compared to these in extraneoplastic mucosa (o2 test; p<0. 0001, and Fisher's exact test; p<0.0001), while the increase in TSP1 expression was not significant. BAI1 expression was slightly decreased in the cancer tissue. These results suggested that specific types of angiostatic factors might have protective roles against cancer cell proliferation via dormancy due to hyponutrition caused by decreased vascularity.

Spinal cord contusion produces a central lesion surrounded by a peripheral rim of residual white matter. Despite stimulation of NG2(+) progenitor cell proliferation, the lesion remains devoid of normal glia chronically after spinal cord injury (SCI). To investigate potential cell-cell interactions of the predominant cells in the lesion at 3 days after injury, we used magnetic activated cell sorting to purify NG2(+) progenitors and OX42(+) microglia/macrophages from contused rat spinal cord. Purified NG2(+) cells from the injured cord grew into spherical masses when cultured in defined medium with FGF2 plus GGF2. The purified OX42(+) cells did not form spheroids and significantly reduced sphere growth by NG2(+) cells in co-cultures. Conditioned medium from these OX42(+) cells, unlike that from normal peritoneal macrophages or astrocytes also inhibited growth of NG2(+) cells, suggesting inhibition by secreted factors. Expression analysis of freshly purified OX42(+) cells for a panel of six genes for secreted factors showed expression of several that could contribute to inhibition of NG2(+) cells. Further, the pattern of expression of four of these, TNFalpha, TSP1, TIMP1, MMP9, in sequential coronal tissue segments from a 2 cm length of cord centered on the injury epicenter correlated with the expression of Iba1, a marker gene for OX42(+) cells, strongly suggesting a potential regional influence by activated microglia/macrophages on NG2(+) cells in vivo after SCI. Thus, the nonreplacement of lost glial cells in the central lesion zone may involve, at least in part, inhibitory factors produced by microglia/macrophages that are concentrated within the lesion.