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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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.

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.

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.

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.

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.

Expression profiling in the muscular dystrophies: identification of novel aspects of molecular pathophysiology.

We used expression profiling to define the pathophysiological cascades involved in the progression of two muscular dystrophies with known primary biochemical defects, dystrophin deficiency (Duchenne muscular dystrophy) and alpha-sarcoglycan deficiency (a dystrophin-associated protein). We employed a novel protocol for expression profiling in human tissues using mixed samples of multiple patients and iterative comparisons of duplicate datasets. We found evidence for both incomplete differentiation of patient muscle, and for dedifferentiation of myofibers to alternative lineages with advancing age. One developmentally regulated gene characterized in detail, alpha-cardiac actin, showed abnormal persistent expression after birth in 60% of Duchenne dystrophy myofibers. The majority of myofibers ( approximately 80%) remained strongly positive for this protein throughout the course of the disease. Other developmentally regulated genes that showed widespread overexpression in these muscular dystrophies included embryonic myosin heavy chain, versican, acetylcholine receptor alpha-1, secreted protein, acidic and rich in cysteine/osteonectin, and thrombospondin 4. We hypothesize that the abnormal Ca(2)+ influx in dystrophin- and alpha-sarcoglycan-deficient myofibers leads to altered developmental programming of developing and regenerating myofibers. The finding of upregulation of HLA-DR and factor XIIIa led to the novel identification of activated dendritic cell infiltration in dystrophic muscle; these cells mediate immune responses and likely induce microenvironmental changes in muscle. We also document a general metabolic crisis in dystrophic muscle, with large scale downregulation of nuclear-encoded mitochondrial gene expression. Finally, our expression profiling results show that primary genetic defects can be identified by a reduction in the corresponding RNA.

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.

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.

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.

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.

Immunohistochemical labelling of components of the endoneurial extracellular matrix of intact and rhizotomized dorsal and ventral spinal roots of the rat--a quantitative evaluation using image analysis.

The endoneurial extracellular matrix (ECM) molecules are involved in cell signalling during nervous system development and regeneration. Quantitative differences of immunofluorescence labelling for chondroitin sulfate proteoglycan (CSPG), fibronectin (FN), tenascin-C (TN-C), and thrombospondin (TSP) were evaluated in intact rat dorsal and ventral roots and dorsal and ventral roots 2 and 4 weeks after rhizotomy using image analysis. The distal stumps of spinal roots displayed increased immunolabelling for the molecules with higher immunofluorescence in dorsal than in ventral roots up to 2 weeks from transection. Four weeks after rhizotomy, the immunoreactivity for CSPG, TN-C and TSP decreased in dorsal and increased in ventral root stumps, although a higher level of immunofluorescence for FN remained in both dorsal and ventral root stumps 4 weeks after injury in comparison to 2 weeks after injury. We suggest that the amount of some ECM molecules changed differentially 2 and 4 weeks after rhizotomy to create an appropriate environment in the endoneurium for early and later regrowth of sensory and motor axons. The results presented here are the first report of differences between the endoneurial ECM content of damaged afferent and motor nerve fibers. In addition, the immunohistochemical detection of individual ECM molecules indicated that final extrinsic conditions stimulating the regrowth of regenerating axons probably arise from a balance of both growth-promoting and -inhibiting molecules in the endoneurium.

Induction of thrombospondin-1 by all-trans retinoic acid modulates growth and differentiation of HL-60 myeloid leukemia cells.

Thrombospondin-1 (TSP), a multifunctional extracellular matrix protein, modulates human hematopoietic stem cell adherence and thus may play a role in blood cell proliferation and/or differentiation. The expression of TSP was studied in the human myeloid leukemia cell line, HL-60, upon differentiation into monocytes by phorbol-13-monoacetate (PMA) or into granulocytes by all-trans retinoic acid (RA). HL-60 cells cultured under serum-free conditions constitutively secreted low amounts of TSP into the cultured medium, approximately 13 ng/10(6) cells/24 h. PMA used at 4 x 10(-8) M did not significantly modulate TSP secretion over a 24 h period. In contrast, RA at 10(-7) M induced a 5- to 10-fold increase in TSP secreted by HL-60 cells during their differentiation into granulocytes over a 5 day period. The role of secreted TSP in RA-dependent cessation of growth and differentiation was examined using blocking anti-TSP antibodies. In the presence of the polyclonal anti-TSP antibody R5 (25 microg/ml), growth of RA-treated HL-60 cells was maintained at control levels for up to 3 days and a concomitant delay in granulocytic differentiation was observed. Moreover, the addition of soluble TSP (0.5-5 microg/ml) to untreated HL-60 cells decreased their growth and promoted their differentiation in a dose-dependent manner. Using a neutralizing antibody to transforming growth factor beta (TGF-beta) or purified TGF-beta1 we further demonstrated that the effects of TSP were not mediated through activation of latent TGF-beta. These studies indicate that TSP decreases the proliferation and promotes the differentiation of HL-60 cells.

Bovine thrombospondin-2: complete complementary deoxyribonucleic acid sequence and immunolocalization in the external zones of the adrenal cortex.

Given the variety of biological functions in the adrenal cortex that are controlled by ACTH, we hypothesized that some extracellular proteins act as biological relays for this systemic hormone. One candidate protein [corticotropin-induced secreted protein (CISP)] was purified from the conditioned medium of bovine adrenocortical cells on the basis of a 5- to 14-fold increase in its synthesis after the addition of ACTH. We report here the cloning of overlapping complementary DNAs that span the sequence encoding the full-length protein (1170 amino acids). The deduced CISP protein sequence is 89% identical to that of human thrombospondin-2 (TSP2), but only 61% identical to that of bovine TSP1, confirming that CISP is the bovine ortholog of TSP2. The bovine TSP2 sequence aligned perfectly with human, mouse, and chicken TSP2 sequences, except for a gap of 2 amino acids located in a linker region. All 58 cysteine residues that are conserved in other species were present in the bovine sequence as well as most of the functional domains. Most endocrine tissues (adrenal cortex, testis, ovary, and placenta) appeared to express TSP2, as determined by Western blot analysis. The highest levels of TSP2 protein were found in the adrenal cortex, followed by the heart, spleen, brain, and kidney. A differential extent of N-glycosylation or tissular proteolytic maturation may be responsible for the mol wt differences observed between bovine TSP2 detected in the medium from primary cultures and that in fresh tissue extracts. The immunohistochemical analysis of the distribution of TSP2 in the bovine adrenal gland revealed that the protein is much more abundant in the external zones (zona glomerulosa and zona fasciculata) than in the internal reticularis zone, a pattern similar to that reported for ACTH receptors. This distribution clearly suggests that TSP2 is a candidate relay protein for a subset of ACTH actions in the adrenal cortex.

Restricted localization of thrombospondin-2 protein during mouse embryogenesis: a comparison to thrombospondin-1.

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

Effect of complement inhibition and heparin coating on artificial surface-induced leukocyte and platelet activation.

BACKGROUND: Exposure of blood to artificial surfaces, as in cardiopulmonary bypass, induces an inflammatory response involving complement, leukocyte and platelet activation. To elucidate the specific role of complement in this process, studies were performed on blood circulated in polyvinyl chloride tubing in the absence and presence of complement inhibitors. Parallel experiments were performed with heparin-coated polyvinyl chloride tubing, which is known to prevent complement and cell activation. METHODS: A novel experimental model was used, based on human whole blood anticoagulated with lepirudin. Complement activation products, myeloperoxidase, lactoferrin, and thrombospondin were quantified in enzyme immunoassays. Leukocyte CD11b expression and leukocyte-platelet conjugates were detected by flow cytometry. RESULTS: Increased levels of C3 activation products, alternative pathway convertase, and the terminal SC5b-9 complex, combined with unchanged levels of C1rs-C1-inhibitor complexes and marginal changes in C4 activation demonstrated that complement was activated through the alternative pathway. Granulocyte and monocyte CD11b expression and granulocyte-platelet conjugate formation were efficiently attenuated by blocking either factor D, C3, C5, or C5a receptor. In contrast, monocyte-platelet conjugate formation and release of myeloperoxidase, lactoferrin, and thrombospondin were not reduced by complement inhibition. Heparin-coated polyvinyl chloride tubing efficiently reduced all inflammatory markers studied, except for C1rs-C1-inhibitor complexes, which increased, consistent with the enhancing effect of heparin on C1-inhibitor function. This effect did not, however, reduce fluid-phase classic pathway activation induced by heat-aggregated immunoglobulin G. CONCLUSIONS: Leukocyte and platelet activation in response to artificial materials occur by mechanisms that vary in their dependence on complement. Heparin coating precludes both the complement-dependent and complement-independent reactions.

Angiogenesis and cigarette smoking in squamous cell lung carcinomas: an immunohistochemical study of 28 cases.

Angiogenesis in tumors is influenced by several factors which in turn are associated with chemoresistance or radioresistance. Moreover, the tumors of smokers are known to be relatively resistant to chemotherapy. This investigation attempts to determine whether or not a relationship exists between cigarette smoking and angiogenesis in lung cancer. Tumor samples from 14 non-smokers and 14 heavy cigarette smokers were selected for this study. The populations were matched for age, sex and tumor stage. Resistance to doxorubicin, microvessel density, the expression of vascular endothelial growth factor (VEGF) and thrombospondin (TSP) were analyzed in both populations. Tumors of smokers were more frequently resistant to doxorubicin in vitro, had lower vessel counts and a reduced expression of VEGF compared to tumors of nonsmokers. In contrast, TSP was significantly increased in the tumors of smokers. These data show that angiogenesis in lung tumors is linked to a patient's smoking habits.