Age-related macular degeneration is a progressive ocular disease that is the leading cause of vision loss among elderly. AMD usually is divided into two types: wet and dry AMD, which is linked with inflammation. Choroidal Neovascularization (CNV) formation or wet AMD is also associated with oxidative stress. Previously, TSP1 has been shown to have a significant alleviating effect on CNV in TSP1 knockout (TSP1^-/-) mice. However, the mechanism by which TSP1 ameliorates CNV remains unclear. Here we report that TSP1 reduces nitric oxide production to prevent cells from forming tubes formation and reduced the levels of vascular endothelial growth factor (VEGF) and lipid peroxides (LPO) during oxidative stress. We measured RF/6A cell viability by CCK-8 assay and apoptosis by flow cytometry. RF/6A cell were transfected with TSP1 and STAT3 overexpression, and then the mRNA and protein levels of TSP1 and also the signal pathways were detected by qRT-PCR and Western blot analysis. Migration assays were performed using a transwell system. Co-Immunoprecipitation was used to analyze the binding relationship between CD47 and SHP-2. The results show that overexpression of TSP1 alleviated the damage of oxidative stress to RF/6A cells including increased cell activity and migration, decreased apoptosis and reduced migration compared to the control group. SHP-2 was activated by TSP1 through its receptor CD47 and STAT3 phosphorylation was reduced by activation of SHP-2, thereby blocking STAT3-iNOS pathway and reducing NO concentration in RF/6A cells ultimately protecting them from oxidative stress. Finally, the CNV mice model confirmed that TSP1 overexpression could protect the mice against CNV in vivo, modified the antioxidants levels and decreased the expression of TNF-α and IL-6 under laser irradiation. These results indicate a potential mechanism of TSP1 to slow down formation of CNV in wet AMD, which may bring hope for new treatment strategies.

CD47 is a ubiquitously expressed transmembrane glycoprotein that plays a complex role in regulation of cell survival and function. We have previously shown that the interspecies incompatibility of CD47 plays an important role in triggering rejection of cellular xenografts by macrophages. However, the role of CD47 in solid organ transplantation remains undetermined. Here, we explored this question in mouse models of heart allotransplantation. We observed that the lack of CD47 in donor hearts had no deleterious effect on graft survival in syngeneic or single MHC class I-mismatched recipients, in which both wild-type (WT) and CD47 knockout (CD47 KO) mouse hearts survived long term with no sign of rejection. Paradoxically, elimination of donor CD47 was beneficial for graft survival in signal MHC class II- and class I- plus class II-mismatched combinations, in which CD47 KO donor hearts showed significantly improved survival compared to WT donor hearts. Similarly, CD47 KO donor hearts were more resistant than WT hearts to humoral rejection in α1,3-galactosyltransferase-deficient mice. Moreover, a significant prolongation of WT allografts was observed in recipient mice treated with antibodies against a CD47 ligand thrombospondin-1 (TSP1) or with TSP1 deficiency, indicating that TSP1-CD47 signaling may stimulate vascularized allograft rejection. Thus, unlike cellular transplantation, donor CD47 expression may accelerate the rejection of vascularized allografts.

The present study aimed to evaluate the role of taxol resistance gene 1 (Txr1) in the development of oxaliplatin (L-OHP) resistance in gastric cancer (GC). Using SGC-7901 cells as a model, Txr1 was exogenously expressed or knocked down using small interfering RNA. Quantitative polymerase chain reaction (qPCR) and western blotting were performed to establish whether the Txr1 gene is involved in chemoresistance, and cell proliferation was assessed using an MTS assay. To this end, the mRNA and protein levels of Txr1, thrombospondin-1 and excision repair cross-complementing 1 protein were measured using qPCR and western blotting, respectively. Txr1-knockdown significantly increased the sensitivity of the SGC-7901 cells to L-OHP, whereas Txr1 overexpression promoted the resistance of the SGC-7901 cells to L-OHP. Exogenous Txr1 expression in the SGC-7901 cells induced L-OHP resistance, and the siRNA knockdown of Txr1 sensitized the human GC cells to L-OHP. Txr1 is, therefore, likely to play a role in L-OHP resistance, acting via TSP1, and should be investigated as a potential therapeutic target in the treatment of GC.

The expression of taxol resistance gene 1 and thrombospondin 1 remains unknown in human breast cancer tissues. In the current study, we sought to measure the mRNA expression levels of taxol resistance gene 1 and thrombospondin 1 in breast cancer tissue and adjacent normal tissue specimens and further analyzed their expression according to the molecular subtypes and age of breast cancer patients who had received taxane-containing regimens. Archived breast cancer and adjacent non-tumor tissue specimens were obtained at Beijing Friendship Hospital, Beijing, China. The mRNA transcript levels of taxol resistance gene 1, thrombospondin 1 and multi-drug resistance 1 were determined by quantitative RT-PCR. Taxol resistance gene 1 and multi-drug resistance 1 protein levels were measured by immunoblotting assays. Forty-nine archived breast cancer tissue specimens were included. The majority of the specimens (65.3%) were of the molecular subtype A followed by triple negative breast cancer (14.3%). The mRNA transcript levels of taxol resistance gene 1 , thrombospondin 1 and multi-drug resistance 1 in breast cancer tissues were higher than those of adjacent normal tissues. The mRNA expression of TXR1 in the HER2 subtype (4.513 ± 0.810) was the highest and in the Luminal B subtype was the lowest (3.103 ± 0.417) among the four molecular subtypes. The HER2 subtype also had the highest mRNA expression of thrombospondin 1(4.827 ± 0.927) and the Luminal B subtype had the lowest TSP1 mRNA level (3.197 ± 0.565) among the four molecular subtypes. Our study represents the first attempt in delineating taxol resistance gene 1 and thrombospondin 1 expression in breast cancer and we demonstrate that taxol resistance gene 1 and thrombospondin 1 expression may vary according to the molecular subtypes of breast cancer.

The purpose of this study was to determine the pathogenic changes that occur in myoepithelial cells (MECs) from lacrimal glands of a mouse model of Sjogren's syndrome. MECs were cultured from lacrimal glands of C57BL/6J (wild type, WT), and thrombospondin 1 null (TSP1^-/-) mice, and from mice expressing smooth muscle actin-GFP (SMA-GFP) that labels MECs. MECs were stimulated with cholinergic and α₁-adrenergic agonists, vasoactive intestinal peptide (VIP), and the purinergic (P)2 agonists, ATP and UTP. Then, intracellular [Ca²⁺] ([Ca²⁺]_i) was measured using fura 2 and contraction was observed using live cell imaging. Expression of purinergic receptors was determined by western blot analysis and mRNA expression was analyzed by microarray. The increase in [Ca²⁺]_i with VIP and UTP was significantly smaller in MECs from TSP1^-/- compared to WT mice. Cholinergic agonists, ATP, and UTP stimulated contraction in MECs although contraction of MECs from TSP1^-/- mice was reduced compared to WT mice. The amount of P2Y1, P2Y11, and P2Y13 was significantly decreased in MECs from TSP1^-/- compared to WT mice whereas several extracellular matrix and inflammation genes were upregulated in MECs from TSP1^-/- mice. We conclude that lacrimal gland MEC function is altered by inflammation as the functions regulated by cholinergic agonists, VIP and purinergic receptors are decreased in TSP1^-/- compared to WT mice.

Keywords: calcium; contraction; dry eye; inflammation; lacrimal gland; myoepithelial cell.

Thrombospondin-1 (TSP1) is involved in corneal wound healing caused by chemical injury. Herein, we examined the effects of TSP1 on hypoxia-induced damages and wound-healing activity in human corneal epithelial (HCE) cells. Exosomal protein expression was determined using liquid chromatography-tandem mass spectrometry, and HCE cell migration and motility were examined through wound-healing assay and time-lapse microscopy. Reestablishment of cell junctions by TSP1 was assessed through confocal microscopy and 3D image reconstruction. Our results show that CoCl₂ -induced hypoxia promoted HCE cell death by paraptosis. TSP1 protected these cells against paraptosis by attenuating mitochondrial membrane potential depletion, swelling and dilation of endoplasmic reticulum and mitochondria, and mitochondrial fission. Exosomes isolated from HCE cells treated with TSP1 contained wound healing-associated proteins that were taken up by HCE cells to promote tissue remodeling and repair. TSP1 protected HCE cells against hypoxia-induced damages and inhibited paraptosis progression by promoting cell migration, cell-cell adhesion, and extracellular matrix remodeling. These findings indicate that TSP1 ameliorates hypoxia-induced paraptosis in HCE cells and promotes wound healing and remodeling by regulating exosomal protein expression. TSP1 may, therefore, play important roles in the treatment of hypoxia-associated corneal diseases.

Keywords: exosomes; human corneal epithelium; hypoxia; paraptosis; thrombospondin-1.

Scar forming wounds are often characterized by higher levels of vascularity than non-scarring wounds and normal skin, and inhibition of angiogenesis has been shown to inhibit scar formation in some model systems. The rabbit ear hypertrophic scar (HS) model has been widely used to study the mechanisms that underlie the development of HS as well as the effectiveness of various treatments. Although the rabbit ear HS model is well characterized in terms of scar formation, the rate and level of angiogenesis has not been investigated in this model, and the cause-effect relationship between angiogenesis and rabbit HSs has not been examined. In the current study, full-thickness excisional wounds were created on the ventral side of New Zealand White rabbit ears to induce HS formation, and the dynamic pattern of angiogenesis and the expression of angiogenic regulatory factors were examined over time. Blood vessel density was found to peak at 2.7% on day 14 post-wounding, decreasing to 1.7% by day 28. mRNA levels of the proangiogenic factor VEGF-A peaked at day 14, while the expression of the antiangiogenic factors pigment epithelium-derived factor (PEDF) and thrombospondin 1 (TSP1) peaked at day 28 post-wounding. To examine whether inhibition of angiogenesis influences HS formation in this model, wounds were treated with exogenous soluble antiangiogenic agents including recombinant PEDF (rPEDF) and a functional PEDF peptide (PEDF-335). rPEDF and PEDF-335 were administered intradermally from day 4 post-wounding every 3 days until day 19. Intradermal injection of rPEDF or PEDF-335 both led to decreased angiogenesis and decreased collagen deposition at the wound site. The results support the utility of antiangiogenic therapies, including rPEDF/PEDF-335, as a potential new strategy for the prevention or treatment of HSs.

The members of the CCN (Cyr61/CTGF/Nov) family are a group of matricellular regulatory proteins that are essential to a wide range of functional pathways in cell signalling. Through interacting with extracellular matrix components and growth factors via one of their four domains, the CCN proteins are involved in critical biological processes such as angiogenesis, cell proliferation, bone development, fibrogenesis and tumorigenesis. Here, the crystal structure of the thrombospondin module 1 (TSP1) domain of CCN3 (previously known as Nov) is presented, which shares a similar three-stranded fold with the thrombospondin type 1 repeats of thrombospondin-1 and spondin-1, but with variations in the disulfide connectivity. Moreover, the CCN3 TSP1 domain lacks the typical π-stacked ladder of charged and aromatic residues on one side of the domain that is seen in other TSP1 domains. Using conservation analysis among orthologous domains, it is shown that a charged cluster in the centre of the domain is the most conserved site and this cluster is predicted to be a potential functional epitope for heparan sulfate binding. This variant TSP1 domain has also been used to revise the sequence determinants of TSP1 domains and to derive improved Pfam sequence profiles for the identification of novel TSP1 domains in more than 10 000 proteins across diverse phyla.

In patients with diabetes, impaired activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13), the plasma metalloprotease that cleaves highly thrombogenic von Willebrand factor multimers, is a major risk factor of cardiovascular events. Here, using Adamts13-/- mice made diabetic by streptozotocin, we investigated the impact of the lack of ADAMTS13 on the development of diabetes-associated end-organ complications. Adamts13-/- mice experienced a shorter life span than their diabetic wild-type littermates. It was surprising that animal death was not related to the occurrence of detectable thrombotic events. The lack of ADAMTS13 drastically increased the propensity for ventricular arrhythmias during dobutamine-induced stress in diabetic mice. Cardiomyocytes of diabetic Adamts13-/- mice exhibited an aberrant distribution of the ventricular gap junction connexin 43 and increased phosphorylation of Ca2+/calmodulin-dependent kinase II (CaMKII), and with the consequent CaMKII-induced disturbance in Ca2+ handling, which underlie propensity for arrhythmia. In vitro, thrombospondin 1 (TSP1) promoted, in a paracrine manner, CaMKII phosphorylation in murine HL-1 cardiomyocytes, and ADAMTS13 acted to inhibit TSP1-induced CaMKII activation. In conclusion, the deficiency of ADAMTS13 may underlie the onset of lethal arrhythmias in diabetes through increased CaMKII phosphorylation in cardiomyocytes. Our findings disclose a novel function for ADAMTS13 beyond its antithrombotic activity.

Background: Several serum biomarkers, including miRNA, mRNA, protein and peptides in cancer patients are also important mediators of cancer progression.

Methods: The differentially expressed peptides between the serum of ovarian cancer patients and healthy controls were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The function of the peptides was analyzed by CCK8, transwell, wound healing, and flow cytometry analysis. And the mechanism of the peptides was analyzed by peptide pull down, and high-throughput RNA-sequencing.

Results: A total of 7 and 46 peptides were significantly up-regulated and down-regulated in the serum of ovarian cancer patients, respectively. The precursor proteins of the differentially expressed peptides mainly involved in the complement and coagulation cascades, platelet activation, phagosome and focal adhesion pathways. Interestingly, focal adhesion, platelet activation, platelet-cancer cell interaction, complement activation, coagulation cascades and phagosome formation are all critical factors for cancer initiation or progression, which indicated that the peptides may play a crucial role in cancer development. And we identified one peptide, ZYX_36-58, which was down-regulated in the serum of ovarian cancer patients, significantly inhibited invasion and migration and promoted the apoptosis of ovarian cancer cells. Mechanistic study indicated that ZYX_36-58 interacted with and increased the protein level of the antiangiogenic protein thrombospondin-1 (TSP1), which has a tumor suppressive effect on ovarian cancer.

Conclusions: ZYX_36-58, which was significantly down-regulated in the serum of ovarian cancer patients can significantly inhibit cell invasion, migration and promote apoptosis of ovarian cancer cells by binding and up-regulating TSP1 protein expression.

Keywords: Peptidome; TSP1; ZYX36-58; migration; ovarian cancer (OC).

OBJECTIVE

To explore the effect of adenovirus-mediated delivery of an antiangiogenic fragment of human thrombospondin 1 (TSP1(f)) on K562 cell growth in nude mice.

METHODS

TSP1(f) cDNA was amplified by RT-PCR from normal human peripheral blood mononuclear cells and was used as transgene to construct a adenoviral vector (ADV-TSP1(f)). Human leukemia K562 cells were cultured and infected with ADV-TSP1(f) or ADV-LacZ. PBS was used as control. TSP1(f) expression/secretion by these infected K562 cells was demonstrated using Western blot analysis. MTT assay was performed to determine the effect of ADV-TSP1(f) infection on K562 cell growth kinetics. Eighteen female Balb/c nude mice were inoculated subcutaneously with human leukemia K562 cells. When the diameter of the tumor reached 5 approximately 7 mm the rats were randomly divided into 3 groups of 6 rats injected intratumorally with ADV-TSP1(f), ADV-LacZ, and PBS respectively. The volume of K562 xenografts was measured every three days during the 3-week treatment. By the end of the 21st day the mice were killed and the tumors were taken to undergo histological examination. The intratumoral microvessel density (MVD) was determined by immunohistochemical staining.

RESULTS

TSP-1f was expressed and secreted efficiently by ADV-TSP1(f)-infected K562 cells. Three weeks after the initial treatment, the volume of K562 xenografts in the mice treated with ADV-TSP1(f), ADV-LacZ, and PBS was (1,108 +/- 179) mm(3), (4,518 +/- 452) mm(3), and (4,666 +/- 458) mm(3) respectively (P < 0.01). The number of CD31+ microvessels counted per x200 field was 34 +/- 9, 36 +/- 7, and 14 +/- 4 in the tumors treated with PBS, ADV.LacZ, and ADV.TSP-1f respectively (P < 0.001).

CONCLUSIONS

Adenovirus-mediated TSP-1f gene transfer greatly inhibits K562-derived tumor growth and angiogenesis in mouse xenograft model, and may serve as a new therapy for hematological malignancies.

The phenomenon of vasculogenic mimicry in melanoma has been recently described to be an important factor relating to melanoma progression. Large scale gene expression profiling by real-time quantitative RT-QPCR of a panel of 40 normal tissues and 54 cancer cell lines revealed that two genetically related melanoma cell lines, one derived from a primary lesion Hs.688(A) and one derived from a lymph node metastasis Hs.688(B), displayed a unique expression pattern when compared to other cancer cell lines and tissue samples in the panel. Quantitative-RT-PCR data indicated that these melanoma cells expressed a number of activated endothelial cell-associated genes such as tissue inhibitors of matrix metalloproteinases TIMP-2, matrix metalloproteinase (MMP-1, MMP-2), thrombospondin 1 (TSP1), proto-oncogene c-MET and vascular endothelial growth factor (VEGF). To examine the gene expression profile of these unique melanoma cells in greater depth, cDNA libraries were made from isolated microsome complexes to enrich those transcripts that were destined to be translated into cell surface or secreted proteins. High throughput sequencing analysis revealed that this library contained over 7000 cDNAs and was enriched by over 80% of secreted or membrane-bound proteins. The presence in the cDNA library of genes such as acetyl LDL receptor, tumor endothelial markers-1, 5 and 8 (TEMs), flow-induced endothelial G protein coupled receptor-1 and VEGF-related protein (VRP), all of which are known to be expressed uniquely by endothelial cells, supported the hypothesis that Hs.688(A) and Hs.688(B) cells were mimicking an activated vascular phenotype. Ultimately the goal is to investigate the biological roles of endothelial cell-associated genes in the behavior of Hs.688(A) and Hs.688 (B) melanoma cells.

The treatment of primary cultures of bovine adrenocortical cells with nanomolar concentrations of ACTH induces a 10-fold increase in the synthesis of a secreted protein of apparent molecular mass 195 kDa on reducing SDS-polyacrylamide gels. This corticotropin-induced secreted protein (CISP) appears to be an oligomeric calcium-binding protein. Its secretion under serum-free culture conditions is sustained over 4 days in the continuous presence of ACTH. Induction of CISP secretion by ACTH is mimicked by cAMP analogs and adenylate cyclase activators. We report here the purification of CISP to apparent homogeneity with an overall yield of 43% using a combination of heparin-agarose and Mono-Q chromatographies. The NH2-terminal amino acid sequence and the sequence of several tryptic peptides revealed that CISP is structurally related to the members of the thrombospondin (TSP) family. Among these members, bovine CISP appeared to be more homologous to mouse TSP2 (85% identity in the 29 amino acid long NH2-terminal sequence) than to TSP1 (18% identity in the same region). We also observed that CISP binds Ca2+ and is an adhesive protein for bovine adrenocortical cells. Thus, CISP possesses both structural and functional properties of thrombospondins. Whether CISP represents the bovine form of TSP2 or a novel member of the expanding thrombospondin family will need to be elucidated by cloning and sequencing of a larger portion of the molecule.

Background: As a common pathological type of glomerular disease in China, mesangial proliferative glomerulonephritis is related to plasminogen activator inhibitor-1 (PAI-1) and thrombospondin-1 (TSP-1). Here, this study aims to investigate the expression and clinical significance of TSP-1 and PAI-1 in patients with mesangial proliferative glomerulonephritis.

Methods: Renal tissue specimens from 46 patients with mesangial proliferative glomerulonephritis admitted to this hospital were selected as the subjects, and 8 specimens of renal tissue from autopsy were used as controls. The expression levels of TSP-1 and PAI-1 were detected by immunohistochemistry and analyzed.

Results: The 24-hour urine protein, triglyceride, and total cholesterol levels of patients with severe mesangial hyperplasia were significantly higher than those of patients with mild and moderate mesangial hyperplasia, and serum albumin was lower than that of patients with mild and moderate mesangial hyperplasia (P<0.05). The 24-hour urine protein level of patients with moderate mesangial hyperplasia was higher than that of patients with mild mesangial hyperplasia while the albumin level was lower (P<0.05), but there was no significant difference in triglyceride and total cholesterol (P>0.05). There was no significant difference in creatinine clearance (Ccr) between the three groups (P>0.05).The 24-hour urine protein and urine alpha-1-microglobulin (A1M) levels in patients with renal interstitial disease were higher than those in patients without renal interstitial disease, while their Ccr level was lower (P<0.05). TSP-1 and PAI-1 were not positively expressed in the glomeruli and renal tubules of specimens of the control group. However, in mesangial hyperplasia patients, the expression of TSP-1 and PAI-1 in mesangial hyperplasia with varying degrees and in different renal tubular damage were as follows: mild degree < moderate degree < severe degree (P<0.05).

Conclusions: The pathological changes of mesangial proliferative glomerulonephritis are related to 24- hour urine protein, triglyceride, total cholesterol level, urine A1M, and Ccr level. The expression of TSP1 and PAI-1 in the mesenchyme of glomerular and renal tubules significantly increased with the severity of the disease, suggesting that TSP-1 and PAI-1 play an important role in the occurrence and development of mesangial proliferative glomerulonephritis.

Keywords: Thrombospondin-1 (TSP-1); immunohistochemistry; mesangial proliferative glomerulonephritis; plasminogen activator inhibitor-1 (PAI-1).

Chronic lung allograft dysfunction (CLAD) is the major cause of death after lung transplantation. Angiotensin II (AngII), the main effector of the renin-angiotensin (RA) system, elicits fibrosis in both kidney and lung. We identified 6 AngII-regulated proteins (RHOB, BST1, LYPA1, GLNA, TSP1, LAMB1) increased in urine of patients with kidney allograft fibrosis. We hypothesized that RA system is active in CLAD and that AngII-regulated proteins are increased in bronchoalveolar lavage fluid (BAL) of CLAD patients.We performed immunostaining of AngII receptors (AGTR1 and AGTR2) and TSP1/GLNA in 10 CLAD lungs and 5 controls. Using mass spectrometry, we quantified peptides corresponding to AngII-regulated proteins in BAL of 40 lung transplant recipients (CLAD, stable and acute lung allograft dysfunction (ALAD)). Machine learning algorithms were developed to predict CLAD based on BAL peptide concentrations.Immunostaining demonstrated significantly more AGTR1+ cells in CLAD versus control lungs (p=0.02). TSP1 and GLNA immunostaining positively correlated with the degree of lung fibrosis (R²=0.42 and 0.57, respectively). In BAL, we noted a trend toward higher concentrations of AngII-regulated peptides in patients with CLAD at the time of bronchoscopy, and significantly higher concentrations of BST1, GLNA and RHOB peptides in patients that developed CLAD at follow-up (p<0.05). Support vector machine classifier discriminated CLAD from stable and ALAD patients at the time of bronchoscopy with AUC 0.86, and accurately predicted subsequent CLAD development (AUC 0.97).Proteins involved in the RA system are increased in CLAD lung and BAL. AngII-regulated peptides measured in BAL may accurately identify patients with CLAD and predict subsequent CLAD development.

Astrocytes respond to environmental cues, including changes in temperatures. Increased deimination, observed in many progressive neurological diseases, is thought to be contributed by astrocytes. We determined the level of deimination and expression of peptidyl arginine deiminase 2 (PAD2) in isolated primary astrocytes in response to changes on either side (31°C and 41°C) of the optimal temperature (37°C). We investigated changes in the astrocytes by using a number of established markers and accounted for cell death with the CellTiter-Blue assay. We found increased expression of glial fibrillary acidic protein, ALDH1L1, and J1-31, resulting from increased incubation temperature and increased expression of TSP1, S100β, and AQP4, resulting from decreased incubation temperature vs. optimal temperature, suggesting activation of different biochemical pathways in astrocytes associated with different incubation temperatures. Mass spectrometric analyses support such trends. The PAD2 level was increased only as a result of increased incubation temperature with a commensurate increased level of deimination. Actin cytoskeleton and iso[4]LGE, a lipid peroxidase modification, also showed an increase with higher incubation temperature. Altogether, these results suggest that temperature, as an environmental cue, activates astrocytes in a different manner on either side of the optimal temperature and that increase in deimination is associated only with the higher temperature side of the spectrum.

Small secreted cysteine-rich proteins (SSCPs) from fungi play an important role in fungi-host interactions. The plant-beneficial fungi Trichoderma spp. are in use worldwide as biocontrol agents and protect the host plant from soil-borne as well as foliar pathogens. Recently, a novel SSCP, Tsp1, has been identified in the secreted protein pool of T. virens and is overinduced upon its interaction with the roots of the maize plant. The protein was observed to be well conserved in the Ascomycota division of fungi, and its homologs are present in many plant-pathogenic fungi such as Fusarium oxysporum and Magnaporthe oryzae. However, none of these homologs have yet been characterized. Recombinant Tsp1 protein has been expressed and purified using an Escherichia coli expression system. The protein, with four conserved cysteines, forms a dimer in solution as observed by size-exclusion chromatography. The dimerization, however, does not involve disulfide bonds. Circular-dichroism data suggested that the protein has a β-strand-rich secondary structure that matched well with the secondary structure predicted using bioinformatics methods. The protein was crystallized using sodium malonate as a precipitant. The crystals diffracted X-rays to 1.7 Å resolution and belonged to the orthorhombic space group P2₁11 (R_meas = 5.4%), with unit-cell parameters a = 46.3, b = 67.0, c = 173.2 Å. The Matthews coefficient (V_M) of the crystal is 2.32 ų Da^-1, which corresponds to nearly 47% solvent content with four subunits of Tsp1 protein in the asymmetric unit. This is the first report of the structural study of any homolog of the novel Tsp1 protein. These structural studies will help in understanding the classification and function of the protein.

Keywords: Trichoderma virens; effector; protein crystallization; secreted cysteine-rich proteins.

Human THROMBOSPONDIN-1 play versatile roles in platelet aggregation, angiogenesis, and tumorigenesis, which forms a disulfide-linked homotrimeric complex. Here we reported that the genomic lotus of TSP1 also transcribes a natural antisense transcript (NAT) with an overlapping and reverse complement region against TSP1. The NAT of TSP1 was expressed in human testis, lung, thymus, colon, placenta, kidney and skeleton muscle, revealed by PCR amplification. It was also expressed in some tumor cell lines. The identification of NAT of TSP1 would be of significant importance to understand the functions of TSP1, and it would also suggest the potential attempt of using RNA interference for related tumor therapy, for instance, breast cancer.

CISP (corticotropin-induced secreted protein) is a secreted protein recently purified in our laboratory from the conditioned medium of ACTH-treated bovine adrenocortical cells. Partial amino acid sequencing of CISP revealed homology with thrombospondins (TSPs), a family of adhesive proteins and in particular with TSP2. We report here the characterization of the molecular structure of CISP. Analysis of CISP by polyacrylamide gel electrophoresis in the absence or presence of SDS indicated an apparent molecular mass approximately equal to 600 kDa for the unreduced protein and an apparent molecular mass of 195 kDa after reduction by 2-mercaptoethanol. The sedimentation coefficient of CISP determined by ultracentrifugation on sucrose gradients was shifted from 9.7 S in the absence to 5.7 S in the presence of 2-mercaptoethanol. These data are consistent with a trimeric organization of the CISP molecule in which 195-kDa monomers would be linked together by disulfide bonds. The trimeric structure of CISP could be observed by rotary shadowing/electron microscopy, where CISP appeared to be composed of three equally electron-dense nodules and of a fourth nodule formed by the close association of three smaller fragments. The overall size of the molecule was 60 nm. We also observed that CISP is sulfated and glycosylated. Using glycosylation inhibitors, we could determine that CISP is synthesized as a 175-kDa core protein, is then matured into a 190-kDa high-mannose form and secreted as a 195-kDa mature protein. Inhibition of sulfation by chlorate did not prevent CISP secretion, whereas inhibition of glycosylation by tunicamycin blocked it. Taken together, these data indicate that the TSP2-related CISP molecule presents both structural and functional properties very similar to those of TSP1. CISP differs greatly, however, from TSP1 by the inducibility of its synthesis by cAMP.

Venous malformation (VM) is a kind of congenital vascular anomaly with high recurrence, and screening for VM lacks an efficient, inexpensive and noninvasive approach now. Serum miRNAs with stable structures are expected to become new postoperative and postablative monitoring biomarkers. Thus, we identified a prognostic serum miR-18a-5p and validated its function in VM. Notably, higher expression level of miR-18a-5p was detected in VM patients than in healthy individuals. We found that miR-18a-5p plays a promotive role in human umbilical vein endothelial cells in vitro. In addition, immunohistochemistry (IHC) results showed a distinct increase of vessels in miR-18a-5p mimics group and a decrease of vessels in inhibitors group compared to the control group in a murine VM model. Furthermore, thrombospondin-1 (TSP1), a potential miR-18a-5p-binding protein, was identified via RNA-seq, luciferase reporter and RNA immunoprecipitation (RIP) assays. Moreover, miR-18a-5p regulated the activation of P53 signaling pathway constituents and consequently led to the regulation of proliferation, migration, invasion and angiogenesis. These results provide a strong theoretical basis for further investigations into pathological mechanism of VM and may provide novel and noninvasive biomarker for VM diagnosis and monitoring.

Keywords: P53; Serum; biomarker; miR-18a-5p; thrombospondin-1; venous malformation.

Tumor angiogenesis is regulated by pro- and anti-angiogenic factors. Anti-angiogenic agents target the interconnected network of angiogenic factors to inhibit neovascularization, which subsequently impedes tumor growth. Due to the complexity of this network, optimizing anti-angiogenic cancer treatments requires detailed knowledge at a systems level. In this study, we constructed a tumor tissue-based model to better understand how the angiogenic network is regulated by opposing mediators at the extracellular level. We consider the network comprised of two pro-angiogenic factors: vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2), and two anti-angiogenic factors: thrombospondin-1 (TSP1) and platelet factor 4 (PF4). The model's prediction of angiogenic factors' distribution in tumor tissue reveals the localization of different factors and indicates the angiogenic state of the tumor. We explored how the distributions are affected by the secretion of the pro- and anti-angiogenic factors, illustrating how the angiogenic network is regulated in the extracellular space. Interestingly, we identified a counterintuitive result that the secretion of the anti-angiogenic factor PF4 can enhance pro-angiogenic signaling by elevating the levels of the interstitial and surface-level pro-angiogenic species. This counterintuitive situation is pertinent to the clinical setting, such as the release of anti-angiogenic factors in platelet activation or the administration of exogenous PF4 for anti-angiogenic therapy. Our study provides mechanistic insights into this counterintuitive result and highlights the role of heparan sulfate proteoglycans in regulating the interactions between angiogenic factors. This work complements previous studies aimed at understanding the formation of angiogenic complexes in tumor tissue and helps in the development of anti-cancer strategies targeting angiogenesis.

Low dose metronomic chemotherapy (MC) is becoming a mainstream treatment for cancer in veterinary medicine. Its mechanism of action is anti-angiogenesis by lowering vascular endothelial growth factor (VEGF) and increasing trombospondin-1 (TSP1). It has also been adopted as a compassionate treatment in very advanced human cancer. However, one of the main limitations of this therapy is its short-term effectiveness: 6 to 12 months, after which resistance develops. pH-centered cancer treatment (pHT) has been proposed as a complementary therapy in cancer, but it has not been adopted or tested as a mainstream protocol, in spite of existing evidence of its advantages and benefits. Many of the factors directly or indirectly involved in MC and anti-angiogenic treatment resistance are appropriately antagonized by pHT. This led to the testing of an association between these two treatments. Preliminary evidence indicates that the association of MC and pHT has the ability to reduce anti-angiogenic treatment limitations and develop synergistic anti-cancer effects. This review will describe each of these treatments and will analyze the fundamentals of their synergy.