Identification of adhesion-associated extracellular matrix component thrombospondin 3 as a prognostic signature for clear cell renal cell carcinoma.

PURPOSE: Clear cell renal cell carcinoma (ccRCC) is a highly aggressive disease, and approximately 30% of patients are diagnosed at the metastatic stage. Even with targeted therapies, the prognosis of advanced ccRCC is poor. The aim of this study was to investigate clinical prognosis signatures by analyzing the ccRCC datasets in The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and the function of thrombospondin 3 (THBS3) in ccRCC. MATERIALS AND METHODS: We analyzed the ccRCC datasets in TCGA and CPTAC to search for extracellular matrix (ECM)-related and adhesion-associated genes, and conducted overall survival, Cox, and receiver operating characteristic analyses. We also performed CCK8, colony formation, and transwell assays to compared the proliferation and migration ability of THBS3 knockout cells with those of cells without THBS3 knockout. RESULTS: Comprehensive bioinformatics analysis revealed that THBS3 is a novel candidate oncogene that is overexpressed in ccRCC tumor tissue and that its elevated expression indicates poor prognosis. Our study also showed that knockdown of THBS3 inhibits proliferation, colony formation, and migration of ccRCC cells. CONCLUSIONS: In summary, our data have revealed that THBS3 is upregulated in cancer tissues and could be used as a novel prognostic marker for ccRCC. Our findings thus offer theoretical support with bioinformatics analyses to the study of ECM and adhesion proteins in ccRCC, which may provide a new perspective for the clinical management of ccRCC.

Sequence and phylogenetic analysis of the thrombospondin-related adhesive protein gene of Babesia gibsoni isolates in dogs in South India.

Babesia gibsoni, the causative agent of canine piroplasmosis, is a tick-borne intraerythrocytic protozoan parasite predominantly reported in Asian countries. The present study aimed at genotypic characterization of B. gibsoni isolates prevalent in dogs in Kerala, a southern state of India. Blood samples were collected from 272 dogs in Kerala and B. gibsoni infection was detected by microscopy and polymerase chain reaction (PCR). Molecular confirmation of B. gibsoni parasites was carried out by 18S rRNA nested-PCR, followed by sequencing. Nested-PCR detected a higher percentage of dogs (40.44%) positive for B. gibsoni infection than microscopy where 15.81% dogs were detected positive for infection. Genetic characterization of B. gibsoni isolates (n = 11) prevalent in dogs in the state of Kerala was carried out by PCR amplification and sequencing of the 855 bp thrombospondin-related adhesive protein (TRAP) gene fragment. Phylogenetic analysis of the B. gibsoni TRAP (BgTRAP) gene revealed that B. gibsoni isolates from Kerala formed a distinct cluster with the isolates from north India and Bangladesh, away from other East Asian isolates. Nucleotide analysis of the tandem repeats of BgTRAP gene showed considerable genetic variation among Indian isolates that was shared by B. gibsoni isolates of Bangladesh but not by the isolates of East Asian countries. The results of the present study further confirmed that B. gibsoni parasites in a distinct genetic clade are endemic in dogs in India and Bangladesh. However, elaborate studies are required for better understanding of the genetic diversity of B. gibsoni.

Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy.

Membranous nephropathy is characterized by deposition of immune complexes along the glomerular basement membrane. PLA2R and THSD7A are target antigens in 70% and 1-5% of primary membranous nephropathy cases, respectively. In the remaining cases, the target antigen is unknown. Here, laser microdissection of glomeruli followed by mass spectrometry was used to identify novel antigen(s) in PLA2R-negative membranous nephropathy. An initial pilot mass spectrometry study in 35 cases of PLA2R-negative membranous nephropathy showed high spectral counts for neural tissue encoding protein with EGF-like repeats, NELL-1, in six cases. Mass spectrometry failed to detect NELL-1 in 23 PLA2R-associated membranous nephropathy and 88 controls. NELL-1 was localized by immunohistochemistry, which showed bright granular glomerular basement membrane staining for NELL-1 in all six cases. Next, an additional 23 NELL-1 positive cases of membranous nephropathy were identified by immunohistochemistry in a discovery cohort of 91 PLA2R-negative membranous nephropathy cases, 14 were confirmed by mass spectrometry. Thus, 29 of 126 PLA2R-negative cases were positive for NELL-1. PLA2R-associated membranous nephropathy and controls stained negative for NELL-1. We then identified five NELL-1 positive cases of membranous nephropathy out of 84 PLA2R and THSD7A-negative cases in two validation cohorts from France and Belgium. By confocal microscopy, both IgG and NELL-1 co-localized to the glomerular basement membrane. Western blot analysis showed reactivity to NELL-1 in five available sera, but no reactivity in control sera. Clinical and biopsy findings of NELL-1 positive membranous nephropathy showed features of primary membranous nephropathy. Thus, a subset of membranous nephropathy is associated with accumulation and co-localization of NELL-1 and IgG along the glomerular basement membrane, and with anti-NELL-1 antibodies in the serum. Hence, NELL-1 defines a distinct type of primary membranous nephropathy.

Membranous nephropathy and thrombotic microangiopathy secondary to metastatic prostate cancer after Iodine-125 prostate brachytherapy: A case report
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BACKGROUND: Prostate cancer is the second most common solid tumor leading to membranous nephropathy (MN). Thrombotic microangiopathy (TMA) has been reported to be related to prostate cancer. Nonetheless, the association between prostate cancer and MN and TMA has not been well established. CASE REPORT: A 73-year-old man presented with nephritic syndrome 40 days after implantation of iodine-125 seed for stage II T2N0M0 prostatic carcinoma. The prostatic-specific antigen (PSA) was normalized, and the tumor disappeared after the initial brachytherapy. The circulating autoantibody to phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain containing 7A (THSD7A) was undetectable. Kidney biopsy revealed MN and TMA in glomerulus. Staining of PLA2R, THSD7A, prostate-specific membrane antigen, and prostate acid phosphatase in glomeruli were all negative. The diagnosis of MN and TMA was made, and a combination of steroid therapy and tacrolimus was prescribed. Two weeks after immunosuppressive treatment with prednisone 30 mg/d and tacrolimus 2 mg/d, the patient achieved partial remission in terms of proteinuria. CONCLUSION: This case study was the first report of MN with TMA as manifestations in patients with prostate cancer after I-125 seeds implantation. We hypothesize that prostate cancer may cause MN and TMA, and the mechanism behind this relationship merits further study.
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Compared staining of the phospholipase A2 receptor in the glomeruli of Chinese adults and children with idiopathic membranous nephropathy.

BACKGROUND: The identification of the M-type phospholipase A2 receptor (PLA2R) is a breakthrough recognized as a major target for adults with idiopathic membranous nephropathy (IMN). However, the role PLA2R played in pediatric patients with IMN, particularly in Chinese, has yet to be determined. METHODS: This retrospective study included 187 adult patients and 38 pediatric patients aged 17 years or younger with biopsy proved IMN. The pediatric cohort consisted of 27 children aged from 1 to 12 years and 11 children aged from 13 to 17. Glomerular expression of PLA2R was analyzed in stored, formalin-fixed, paraffin-embedded kidney biopsy sections. RESULTS: PLA2R staining in glomerular deposits was observed in 82.7% and 42.1% of adult and pediatric patients with IMN, respectively. The PLA2R-positive staining patients with IMN presented with more severe clinical features than PLA2R-negative staining patients in both adult and pediatric cohorts. When compared to the young children patients with IMN, the adolescents exhibited a higher positive rate of PLA2R staining (81.8% versus 25.9%), similar to the adult patients. CONCLUSION: The clinical features and prevalence of PLA2R positive staining in adolescent patients with IMN were similar to adult patients, suggesting that they probably have a close etiology and pathogenesis. However, most of the young children patients with IMN were PLA2R negative staining, suggesting a different underlying etiology.

Effects of proteome changes on the tenderness of yak rumen smooth muscle during postmortem storage based on the label-free mass spectrometry.

A label-free proteomics method was used to explore the effects of differentially expressed proteins on the tenderness of yak rumen smooth muscle during postmortem storage (0, 3 and 7 days) at 3 ±â€¯1 °C. The tenderness improved significantly during storage. A total of 212 differentially expressed proteins were identified by the following comparisons: Day 3 vs.0, day 7 vs.0, and day 7 vs.3. Twenty-eight proteins were correlated with the WBSF of yak rumen smooth muscle. Calpastatin, ADP/ATP translocase 1, zyxin, LMOD1 protein, tropomyosin α-3 chain, thrombospondin-4 and UQCRC1 protein are highly related to smooth muscle tenderness, and thus, they are candidates indicators of yak rumen smooth muscle tenderness during storage. Furthermore, bioinformatics analyses revealed that the identified proteins were related to focal adhesion, vascular smooth muscle contraction, cardiac muscle contraction and necroptosis. The present results could provide proteomic insights into changes in yak rumen smooth muscle tenderness during storage and may be a valuable resource for future investigations.

Targeted matrisome analysis identifies thrombospondin-2 and tenascin-C in aligned collagen stroma from invasive breast carcinoma.

Increasing evidence demonstrates an important role for the extracellular matrix (ECM) in breast cancer progression. Collagen type I, a core constituent of the fibrous ECM, undergoes a significant set of changes that accompany tumor progression, termed Tumor Associated Collagen Signatures (TACS). Late stages of this progression are characterized by the presence of bundled, straight collagen (TACS-2) that become oriented perpendicular to the tumor-stromal boundary (TACS-3). Importantly, the presence of TACS-3 collagen is an independent predictor of poor patient outcome. At present, it remains unclear whether reorganization of the collagen matrix is the consequence of mechanical or compositional tissue remodeling. Here, we identify compositional changes in ECM correlating to collagen fiber reorganization from nineteen normal and invasive ductal carcinoma (IDC) patient biopsies using matrisome-targeted proteomics. Twenty-seven ECM proteins were significantly altered in IDC samples compared to normal tissue. Further, a set of nineteen matrisome proteins positively correlate and five proteins inversely correlate with IDC tissues containing straightened collagen fibers. Tenascin-C and thrombospondin-2 significantly co-localized with aligned collagen fibers in IDC tissues. This study highlights the compositional change in matrisome proteins accompanying collagen re-organization during breast cancer progression and provides candidate proteins for investigation into cellular and structural influences on collagen alignment.

Translational Aspects of Primary Membranous Nephropathy.

Membranous nephropathy (MN) in an autoimmune disease caused by binding of circulating antibodies to podocytic antigens. The search for the responsible target antigens has extended for more than 50 years and led to the identification of the major pathomechanisms leading to MN. The combination of clinical and morphologic observations, experimental work, and technical advancements has enabled us deep insights in the pathophysiology of this disease, simultaneously improving treatment of patients. MN represents a perfect example of how patient care may profit from the convergence of scientific and clinical achievements and the benefits of translational approaches in medicine.

Riboflavin and ultraviolet illumination affects selected platelet mRNA transcript amounts differently.

BACKGROUND: Pathogen inactivation (PI) techniques use ultraviolet (UV) illumination with or without a photosensitizer to destroy pathogen RNA and DNA. Although lacking a nucleus and innate DNA transcription, platelets (PLTs) contain RNA and can synthesize proteins. The impact of PI on PLT protein synthesis and function is unknown; altered synthesis may affect overall PLT quality. In this study we determine to what extent PLT RNA is affected by PI. STUDY DESIGN AND METHODS: In a pool-and-split design, paired apheresis PLT concentrates were treated with riboflavin and UV illumination or were left untreated. PLT total RNA and mRNA amounts specific for glycoproteins (GP)IIIa, GPIIb, and GPIb; α-granule proteins PLT factor (PF)4; osteonectin and thrombospondin (TSP); and housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using absorbance and quantitative polymerase chain reaction. RESULTS: After treatment, amounts of all analyzed mRNAs were significantly reduced (p < 0.05), but to different degrees. For GAPDH and PF4, transcripts appeared less susceptible to the treatment, with 70% remaining 1 hour after UV illumination. For GPIIIa and TSP, less than 15% remained after treatment. There was a correlation (R(2) = 0.85) between transcript length and amount of mRNA remaining 1 hour after treatment. Total RNA demonstrated a life span equal to the PLT life span of 10 to 11 days. CONCLUSION: This is the first report of the impact of riboflavin and UV illumination on PLT mRNA. Results suggest that all mRNA present in PLTs is affected by the treatment although the degree of the effect varies among transcripts.

Thrombospondin-4 expression is activated during the stromal response to invasive breast cancer.

The thromobospondins are a family of extracellular glycoproteins that are activated during tissue remodeling processes such as embryogenesis, wound healing and cancer. Thrombospondin-4 (THBS4) is known to have roles in cellular migration, adhesion and attachment, as well as proliferation in different contexts. Data to support a role in cancer biology is increasing, including for gastrointestinal and prostate tumours. Here, using a combination of immunohistochemistry, immunofluorescence and analysis of publicly available genomic and expression data, we present the first study describing the pattern of expression of THBS4 in normal breast and breast cancer. THBS4 was located to the basement membrane of large ducts and vessels in normal breast tissue, but was absent from epithelium and extracellular matrix. There was a significant induction in expression in cancer-associated stroma relative to normal stroma (P = 0.0033), neoplastic epithelium (P < 0.0001) and normal epithelium (P < 0.0001). There was no difference in stromal expression of THBS4 between invasive ductal carcinomas (IDC) and invasive lobular carcinomas (ILC). The THBS4 mRNA levels were variable yet were generally highest in tumours typically rich in stromal content (ILC, ER positive low grade IDC; luminal A and normal-like subtypes). Genomic alterations of the THBS4 gene (somatic mutations and gene copy number) are rare suggesting this dramatic activation in expression is most likely dynamically regulated through the interaction between invading tumour cells and stromal fibroblasts in the local microenvironment. In summary, THBS4 expression in breast cancer-associated extracellular matrix contributes to the activated stromal response exhibited during tumour progression and this may facilitate invasion of tumour cells.

A thrombospondin structural repeat containing rhoptry protein from Plasmodium falciparum mediates erythrocyte invasion.

Host cell invasion by Plasmodium falciparum requires multiple molecular interactions between host receptors and parasite ligands. A family of parasite proteins, which contain the conserved thrombospondin structural repeat motif (TSR), has been implicated in receptor binding during invasion. In this study we have characterized the functional role of a TSR containing blood stage protein referred to as P. falciparum thrombospondin related apical merozoite protein (PfTRAMP). Both native and recombinant PfTRAMP bind untreated as well as neuraminidase, trypsin or chymotrypsin-treated human erythrocytes. PfTRAMP is localized in the rhoptry bulb and is secreted during invasion. Adhesion of microneme protein EBA175 with its erythrocyte receptor glycophorin A provides the signal that triggers release of PfTRAMP from the rhoptries. Rabbit antibodies raised against PfTRAMP block erythrocyte invasion by P. falciparum suggesting that PfTRAMP plays an important functional role in invasion. Combination of antibodies against PfTRAMP with antibodies against microneme protein EBA175 provides an additive inhibitory effect against invasion. These observations suggest that targeting multiple conserved parasite ligands involved in different steps of invasion may provide an effective strategy for the development of vaccines against blood stage malaria parasites.

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

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

Increased thrombospondin-2 in human fibrosclerotic and stenotic aortic valves.

BACKGROUND: Active involvement of extracellular matrix (ECM) and its composition regulating factors may have a central role in the pathogenesis of calcific aortic valve disease (CAVD). Thrombospondins (TSPs) are highly conserved matricellular proteins regulating inflammation, angiogenesis and ECM remodeling. These processes are strongly associated with progression of aortic valve stenosis (AS). However, the expression of TSPs in CAVD is not known. METHODS: We characterized the expression of TSPs 1-4 in human aortic valves by real-time quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Control valves (n=8), thickened and stiffened fibro(sclero)tic valves (n=8), and calcified AS valves (n=24) were compared. Furthermore, potential factors regulating TSP-2 expression was studied by western blotting and gel mobility shift assay in another set of control (n=10) and AS (n=20) valves. RESULTS: TSP-2 mRNA levels were increased 4.9-fold (P=0.037) and 4.8-fold (P=0.001) in fibro(sclero)tic and stenotic valves, respectively, whereas the expression of other TSPs did not change significantly. All TSPs 1-4 were detected from aortic valves by immunohistochemistry. Positive TSP-2 immunostaining was seen in the valvular myofibroblasts and patchily in endothelial cells. Semiquantitative analysis of TSP-2 staining indicated increased immunoreactivity for TSP-2 in neo vessels of fibro(sclero)tic and calcified aortic valves. Finally, when compared to controls, AS was associated with significant down regulation of Akt-pathway and diminished binding activity of nuclear factor-κB (NF-κB). CONCLUSIONS: We report for the first time that TSPs 1-4 are expressed in human aortic valves. CAVD is characterized by myofibroblastic proliferation and neovascularization associated upregulation of TSP-2 expression, as well as inactivation of Akt and NF-κB.

Differential expression of pancreatitis-associated protein and thrombospondins in arterial versus venous tissues.

BACKGROUND/AIMS: Arteries and veins modulate cardiovascular homeostasis and contribute to hypertension pathogenesis. Functional differences between arteries and veins are based upon differences in gene expression. To better characterize these expression patterns, and to identify candidate genes that could be manipulated selectively in the venous system, we performed whole genome expression profiling of arteries and veins. METHODS: We used the CodeLink platform and the major artery (thoracic aorta) and vein (caudal vena cava) of the rat. RESULTS: The most prominent difference was pancreatitis-associated protein (PAP1), expressed 64-fold higher in vena cava versus aorta. Expression of mRNA for thrombospondins (TSP-1, TSP-4) was greater than 5-fold higher in veins versus arteries. Higher mRNA expression of TSP-1, TSP-2, TSP-4 and PAP1 in vena cava versus aorta was confirmed by PCR. Immunohistochemical analysis of tissue sections qualitatively confirmed a higher expression of these proteins in vena cava versus aorta. CONCLUSION: This is the first gene array study of adult rat arterial and venous tissues, and also the first study to report differences in inflammatory genes between arteries and veins. Data from these studies may provide novel insights into the genetic basis for functional differences between arteries and veins in health and disease.

Cancerous, but not stromal, thrombospondin-2 contributes prognosis in pulmonary adenocarcinoma.

Thrombospondin (TSP)-2 is known to be an endogenous negative regulator of vascularization in human cancer. However, it is unclear whether TSP-2 expression is related to neovascularization and prognosis in non-small cell lung cancer. In this study, we quantitatively examined the expression of TSP-2 mRNA by real-time reverse transcription-polymerase chain reaction (RT-PCR) in 102 pulmonary adenocarcinomas. All 102 carcinoma specimens expressed TSP-2 mRNA. The expression of TSP-2 mRNA in carcinoma was significantly higher than normal lung tissues (p<0.0001, Kruskal-Wallis test). Sizes of tumors were significantly correlated with TSP-2 gene expression (p=0.0179, Kruskal-Wallis test). The TSP-2 expression levels of the stage II/III pulmonary carcinomas were significantly increased as compared to those of stage I (p=0.0136, Kruskal-Wallis test). Thirty-five patients with high TSP-2 mRNA expression showed poor prognosis in survival (p=0.0139, log-rank test). We examined TSP-2 protein localizations in the pulmonary adenocarcinoma overexpressing TSP-2 mRNA. The TSP-2 localizations were categorized in two patterns: cancerous TSP-2 expression pattern (TSP-2 expression in the cancerous cells) and non-cancerous TSP-2 expression pattern (TSP-2 expression in the stromal lymphoid cells). Pulmonary adenocarcinoma patients with cancerous TSP-2 expression pattern showed good prognosis (p=0.0322; Fisher's probability exact test). Pulmonary adenocarcinoma patients with non-cancerous TSP-2 expression pattern showed poor prognosis (p=0.0220; Fisher's probability exact test). Non-cancerous TSP-2 expressions may reflect secondary reactions in the cancerous stroma. The stromal TSP-2 expression is not enough to suppress growth of pulmonary adenocarcinoma, while the cancerous TSP-2 expression directly inhibits growth of the carcinoma.

Ultrastructural immunolocalization of cartilage oligomeric matrix protein, thrombospondin-4, and collagen fibril size in rodent achilles tendon in relation to exercise.

Fourteen 3-week-old Sprague-Dawley rats were housed in pairs in standard cages (5 controls) and in individual cages with a running wheel. Four of these rats had run 27-36 km/week (low training - LT) and 5 had run 56-92 km/week (high training - HT). After 4 weeks, the rats were euthanized and Achilles tendons were fixed for electron microscopy. The ultrastructural distribution of cartilage oligomeric matrix protein (COMP) and thrombospondin (TSP)-4 and collagen fibril thickness in two different extracellular compartments were studied. The immunolabeling of COMP decreased with longer running distance and was significantly lower in both the pericellular (p = 0.009) and interterritorial (p = 0.03) compartments of the HT rats compared with the controls. TSP-4 immunolabeling was higher in the pericellular compared with the interterritorial compartments in all rats (p = 0.013) but was not correlated with COMP immunolabeling. No alterations in collagen fibril size were found in relation to running; however, the gold markers representing COMP and TSP-4 were mostly found at the dark bands, representing the gap region of the fibril.

Immunohistochemical identification and localization of endogenous endostatin and its related peptides in murine tumors.

Endostatin, a fragment of the C-terminal domain of mouse collagen XVIII, is a recently demonstrated endogenous inhibitor of tumor angiogenesis. Although endostatin can be detected in blood and urine of tumor-bearing as well as normal mice, the exact localization of the endogenous protein and its related peptides in tumor tissues is unknown. We used immunohistochemistry and immunoblotting to identify endostatin tissue location and staining patterns in tumor, as well as to determine the differences in the levels of endostatin expression between tumor cells (in vitro) and tumor tissues (in vivo). Using a specific polyclonal antibody against murine endostatin, we quantitatively determined the levels of endostatin in five murine mammary tumors and the KHT sarcoma by Western blotting. The staining patterns for this protein in tumor sections were examined histologically by immunohistochemistry. Our results show that: (1) Endogenous endostatin and its related peptides are widely distributed in all in vivo tumor types tested, but not in most of the cultured tumor cell lines. (2) Endogenous endostatin stained most tumor stromal components, including vessel walls, basement membranes, extracellular spaces, and tumor cells. (3) Staining patterns and localization of endostatin and thrombospondin-1 were similar in these tumor sections.

AlphaPS2 integrin-mediated muscle attachment in Drosophila requires the ECM protein Thrombospondin.

During Drosophila embryogenesis, the attachment of somatic muscles to epidermal tendon cells requires heterodimeric PS-integrin proteins (alpha- and beta-subunits). The alpha-subunits are expressed complementarily, either tendon cell- or muscle-specific, whereas the beta-integrin subunit is expressed in both tissues. Mutations of beta-integrin cause a severe muscle detachment phenotype, whereas alpha-subunit mutations have weaker or only larval muscle detachment phenotypes. Furthermore, mutations of extracellular matrix (ECM) proteins known to act as integrin binding partners have comparatively weak effects only, suggesting the presence of additional integrin binding ECM proteins required for proper muscle attachment. Here, we report that mutations in the Drosophila gene thrombospondin (tsp) cause embryonic muscle detachment. tsp is specifically expressed in both developing and mature epidermal tendon cells. Its initial expression in segment border cells, the tendon precursors, is under the control of hedgehog-dependent signaling, whereas tsp expression in differentiated tendon cells depends on the transcription factor encoded by stripe. In the absence of tsp activity, no aspect of muscle pattern formation as well as the initial contact between muscle and tendon cells nor muscle-to-muscle attachments are affected. However, when muscle contractions occur during late embryogenesis, muscles detach from the tendon cells. The Tsp protein is localized to the tendon cell ECM where muscles attach. Genetic interaction studies indicate that Tsp specifically interacts with the alphaPS2 integrin and that this interaction is needed to withstand the forces of muscle contractions at the tendon cells.

Thrombospondin-4 and matrix three-dimensionality in axon outgrowth and adhesion in the developing retina.

Thrombospondin-4 (TSP-4), a large pentameric glycoprotein of the extracellular matrix, has been described as a neurite outgrowth-promoting molecule. However, the means by which TSP-4 promotes neurite outgrowth in the developing eye is unclear. Here we show that TSP-4 is present at the appropriate time in development and displays a localization pattern within the developing mouse retina consistent with a role in retinal ganglion cell (RGC) neurite outgrowth. Furthermore, results indicate that while TSP-4 alone does not support adhesion or neurite extension, it enhances the ability of laminins to promote adhesion and neurite outgrowth of embryonic retinal cells. The mechanism of enhancement is, in part, based on the ability of TSP-4 to enhance the three-dimensionality and/or clustering of laminins within the substrate matrix. These results support a model where TSP-4 acts as an organizer of adhesive and axon outgrowth-promoting molecules in the ECM to optimize retinal ganglion cell responses.

Thrombospondin-1 and thrombospondin-2 mRNA and TSP-1 and TSP-2 protein expression in uterine fibroids and correlation to the genes COL1A1 and COL3A1 and to the collagen cross-link hydroxyproline.

Uterine fibroids are composed of altered collagen fibrils and represent an arrested response to injury-initiating fibrosis. In many tissues, TSP-1 is secreted by adult macrophages and monocytes upon wounding and is involved in the activation of transforming growth factor beta. In the absence of TSP-1, the orchestrated process of wound healing is impaired. The authors obtained tissue from the edge and center of fibroids at the time of hysterectomy and compared them with adjacent myometrium. The pattern of TSP-1 and TSP-2 expression was correlated to that of COL1A1 and COL3A1. Collagen and hydroxyproline were increased in fibroids. Thrombospondin-1 was consistently underexpressed in both the edge and center of the fibroids, while COL1A1 and COL3A1 were consistently overexpressed. However, TSP-2 was inconsistently expressed. These findings lead to the conclusion that the underexpression of TSP-1 may contribute to the overall development of uterine fibroids.