Metastases from uveal melanoma, the most common primary malignant eye tumour in adults, develop solely via their vascular bed due to the absence of intraocular lymphatics. The present study investigated the expression in this tumour of three matricellular proteins--Secreted Protein Acidic and Rich in Cysteine (SPARC), thrombospondin 1 (TSP1) and thrombospondin 2 (TSP2)--with putative contrasting roles in the regulation of angiogenesis. Immunohistochemical analysis of the three proteins was carried out in paraffin-embedded specimens from 27 posterior uveal melanomas and was corroborated with Western blot analysis of fresh-frozen samples from seven of the tumours. SPARC immunoreactivity was detected in all specimens and defined two categories of tumour: SPARC-rich (21 of 27 specimens) and SPARC-patchy (six of 27 specimens) uveal melanomas. SPARC-rich tumours had a significantly higher proportion of specimen area occupied by blood vessels (P=0.04) and showed a positive association with the presence of epithelioid-type tumoral cells (P=0.101). TSP1 was not detected by either of the methods in any of the tumours analysed. Some immunopositivity for TSP2 was detected in tumour cells in approximately 40% of specimens, but was not associated with survival, tumour vascularity or any other histopathological indices of survival. The pattern of expression of these matricellular proteins in uveal melanoma is consistent with a cooperative mechanism for establishing an enhanced environment favourable to angiogenesis. Interventions inducing TSP1 expression and/or inhibiting SPARC expression may be candidates for therapies directed towards the inhibition of angiogenesis in posterior uveal melanoma.

The integrin alpha3beta1 plays important roles in development, angiogenesis, and the pathogenesis of cancer, suggesting potential therapeutic uses for antagonists of this receptor. Recently, an alpha3beta1 integrin-binding site was mapped to residues 190-201 (FQGVLQNVRFVF) of the N-terminal domain of the secreted protein thrombospondin-1 (TSP1). This sequence displays diverse biological activities in vitro and inhibits angiogenesis in vivo. Herein we describe the NMR solution conformation of this segment in both water and dodecylphosphocholine micelles. While essentially unstructured in water, a more well-defined conformation is populated in micelles, particularly in the C-terminal half of the peptide and correlated with increased biological activity of the micellar peptide. The data suggested that the residues that are critical for biological activity are contained in a structurally well-defined segment of the peptide. These data support the role of the NVR motif as a required element of full-length TSP1 for specific molecular recognition by the alpha3beta1 integrin.

BACKGROUND

von Willebrand factor (VWF) cleavage by ADAMTS13 is mediated by multi-step interactions between their multi-domain structures. To clarify the relationship between inhibitory effects of monoclonal antibodies and epitopes on each ADAMTS13 domain, we analyzed how each ADAMTS13 domain contributes to catalyze VWF using a mouse anti-ADAMTS13 monoclonal antibody panel.

METHODS

FRETS-VWF73 assay was used to examine the effects of 14 anti-ADAMTS13 monoclonal antibodies on the catalytic activity of plasma ADAMTS13. Epitope mapping was performed using phage surface display. Libraries expressing peptide fragments of ADAMTS13 were screened with the monoclonal antibodies.

RESULTS

Eleven epitopes of 14 monoclonal antibodies were successfully defined. Three monoclonal antibodies recognizing metalloprotease or disintegrin-like domains strongly inhibited the catalytic activity and their epitopes were on Gln159-Asp166, Tyr 305-Glu327, and Asn308-Glu376. Five monoclonal antibodies recognizing TSP1-3 to -7 repeats showed weak inhibitory effects, and their epitopes were on Pro744-Ala806, Pro856-Cys864, Gln892-Gly940, Cys1007-Cys1072, and Gln1163-Asn1185. Four monoclonal antibodies recognizing the TSP1-1, TSP1-2, CUB1 or CUB2 domains had no inhibitory effects, and their epitopes, except that for TSP1-1, were Pro682-Cys742, Thr1200-Cys1213, and Gln1409-Glu1414. Two monoclonal antibodies recognizing cysteine-rich and spacer domains showed moderate inhibitory effects, but their epitopes were not determined.

CONCLUSIONS

We revealed the epitopes of 11 monoclonal anti-ADAMTS13 antibodies on each of the domains and clarified their association with inhibitory effects on VWF catalysis under static conditions. Catalytic activity correlated strongly with the epitopes on metalloprotease and disintegrin-like domains, weakly with those on TSP1-3 to -7 repeats, and negatively with those on TSP1-1, -2, and CUB domains.

Anthocyanins are known to have antioxidant and antiinflammatory effects. We hypothesized that anthocyanins would enhance wound healing in Sprague-Dawley rats. The purpose of this study was to evaluate our hypothesis and investigate the mechanism of wound healing enhancement. The cytoprotective effect of an immortalized epidermal keratinocyte cell line (HaCaT) and human neonatal dermal fibroblasts in response to various concentrations of anthocyanins was determined. Vascular endothelial growth factor (VEGF) and thrombospondin 1 (TSP1) of HaCaT were measured by Western blot analysis. Anthocyanins were applied to the wounds in rats, and the healing ratio was calculated. Tissue VEGF, TSP1, CD31, nuclear factor-κB, and phosphorylation of IκBα were measured. The viability of the HaCaT cell line and human neonatal dermal fibroblasts increased under cytotoxicity by H2O2 in the anthocyanin-treated groups. The VEGF in the anthocyanin-treated groups increased, whereas TSP1 decreased. Wounds in the experimental groups healed faster, and VEGF and CD31 increased in the experimental groups, whereas TSP1 decreased. Anthocyanins inhibited the translocation of nuclear factor-κB (p65) from cytosol to nucleus and also prevented the phosphorylation of IκBα. Anthocyanins enhance wound healing through a cytoprotective effect, enhancement of angiogenesis, and an antiinflammatory effect.

Thrombospondin-1 (TSP1) is one of the extracellular matrix glycoproteins that affect cell adhesion, motility and growth. Based on its effects on tumors, TSP1 is thought to be a potential regulator of tumor growth and metastasis. In this study, we examined TSP1 expression in human gallbladder adenocarcinoma and its clinicopathological significance. TSP1 immunoreactivity was detected mainly in the cancer stroma and was observed infrequently in cancer cells. According to the TNM classification, 74.5% (29/39) of the T2 and T3 gallbladder cancers were TSP1-positive, while none (0/14) of the T1 cancers showed TSP1 expression (p<0.001). Lymph node metastasis and venous involvement were frequently found in the TSP1-positive cases (90.0% and 87.1%, respectively) of gallbladder adenocarcinoma (p<0.001). These observations suggested that TSP1 expression plays an important role in cancer cell growth and metastasis of human gallbladder adenocarcinomas, and that stromal TSP1 immunoreactivity is a good predictor of vascular involvement and lymph node metastasis.

Thrombospondin-1 (TSP1) is correlated with carcinogenesis occurring in cases of intestinal inflammation. The aim of this study was to clarify the role of TSP1 in gastric carcinogenesis.

A total of 39 patients with gastric cancer who had undergone gastrectomy were enrolled. The expression of TSP1 mRNA in non-cancer tissues was determined. Furthermore, the expression of CD36, STAT3 and TGFβR2 mRNA in non-cancer tissues in two expression groups, the TSP1 high- and low-expression groups, were examined.

The expression of TSP1 was high in the mucosal-atrophy group and tended to be high in the Helicobacter pylori (H. pylori) (+) and multiple cancer groups. The levels of CD36, STAT3 and TGFβR2 mRNA were significantly higher in the TSP1-high group. TSP1 signaling pathway was induced in multiple cancer or atrophy (+) or H. pylori (+) compared to cases with single cancer, atrophy (-) and H. pylori (-). Expression of proteins involved in the TSP1 signaling pathway in non-cancer tissues with multiple gastric cancers were higher than that with single gastric cancer.

Expression of TSP1 in non-cancer tissue correlated with gastric carcinogenesis.

The gastroretentive dosage form of famotidine was modified using tamarind seed powders to prolong the gastric retention time. Tamarind seeds were used in two different forms having different swelling and gelling properties: with husk (TSP) or without husk (TKP). TKP (TKP1 to TKP 6) and TSP (TSP1 to TSP 6) series were prepared using tamarind powder:xanthan in the ratios of 5:0, 4:1, 3:2, 2:3, 1:4, 0:5, respectively. The matrix tablets were prepared by the wet granulation method and evaluated for pharmacopoeial requirements. TKP2 was the optimum formulation as it had a short floating lag time (FLT<30 s) and more than 98.5% drug release in 12 h. The dissolution data were fitted to popular mathematical models to assess the mechanism of drug release, and the optimum formulation showed a predominant first order release and diffusion mechanism. It was concluded that the TKP2 prepared using tamarind kernel powder:xanthan (4:1) was the optimum formulation with shortest floating lag time and more than 90% release in the determined period of time.

Platelets and their granular contents influence both angiogenesis and breast cancer progression. This study was performed to assess the effect of breast cancer and its treatment on platelet biology and the response to inhibition of the platelet P2Y12 receptor. Receptor-specific platelet activation and inhibition was studied for three platelet-associated proteins important in cancer angiogenesis and progression, vascular endothelial growth factor (VEGF), thrombospondin1 (TSP1), and transforming growth factor beta 1 (TGF-β1). Twenty-four women with active breast cancer and 10 healthy controls not receiving antiplatelet therapy participated in the study. Ex vivo activation of platelets in whole blood was accomplished using PAR1AP, PAR4AP, convulxin, and ADP. Platelet inhibition was accomplished using the P2Y12 receptor antagonist cangrelor (the in vitro equivalent of clopidogrel). VEGF, TSP1, and TGF-β1 were measured using standard ELISA. Platelet activation by ADP, PAR1, PAR4, and collagen receptors increased VEGF, TSP1, and TGF-β1 secretion in patients with breast cancer. Agonist-induced release of VEGF was greater in cancer patients as compared to healthy controls (p = 0.02 for ADP, p < 0.001 for PAR1AP, PAR4AP, and convulxin) despite a decrease in the efficiency of VEGF secretion in patients with breast cancer. These differences were not observed for TSP1 and TGF-β1 secretion. P2Y12 receptor inhibition decreased VEGF, TSP1, and TGF-β1 secretion. In patients with cancer, cangrelor inhibited TSP1 release to a greater extent than VEGF and TGF-β1 release. In patients with breast cancer, the magnitude of platelet inhibition achieved by cangrelor was greater than that achieved with healthy controls for all agonists and platelet proteins studied. While platelets are known to influence progression of breast cancer, our results show that breast cancer and its treatment influence the platelet phenotype by increasing the secretion of pro-angiogenic proteins following platelet activation, modulating the efficiency of platelet protein release as well as increasing the response to antiplatelet therapy.

CD47 is an immune checkpoint molecule that downregulates key aspects of both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα, and it is expressed at high levels in a wide variety of tumor types. This has led to the development of biologics that inhibit SIRPα engagement including humanized CD47 antibodies and a soluble SIRPα decoy receptor that are currently undergoing clinical trials. Unfortunately, toxicological issues, including anemia related to on-target mechanisms, are barriers to their clinical advancement. Another potential issue with large biologics that bind CD47 is perturbation of CD47 signaling through its high-affinity interaction with the matricellular protein thrombospondin-1 (TSP1). One approach to avoid these shortcomings is to identify and develop small molecule molecular probes and pretherapeutic agents that would (1) selectively target SIRPα or TSP1 interactions with CD47, (2) provide a route to optimize pharmacokinetics, reduce on-target toxicity and maximize tissue penetration, and (3) allow more flexible routes of administration. As the first step toward this goal, we report the development of an automated quantitative high-throughput screening (qHTS) assay platform capable of screening large diverse drug-like chemical libraries to discover novel small molecules that inhibit CD47-SIRPα interaction. Using time-resolved Förster resonance energy transfer (TR-FRET) and bead-based luminescent oxygen channeling assay formats (AlphaScreen), we developed biochemical assays, optimized their performance, and individually tested them in small-molecule library screening. Based on performance and low false positive rate, the LANCE TR-FRET assay was employed in a ~90,000 compound library qHTS, while the AlphaScreen oxygen channeling assay served as a cross-validation orthogonal assay for follow-up characterization. With this multi-assay strategy, we successfully eliminated compounds that interfered with the assays and identified five compounds that inhibit the CD47-SIRPα interaction; these compounds will be further characterized and later disclosed. Importantly, our results validate the large library qHTS for antagonists of CD47-SIRPα interaction and suggest broad applicability of this approach to screen chemical libraries for other protein-protein interaction modulators.

BACKGROUND

Thrombospondins (TSPs) are recognized as important glycoproteins that regulate a wide variety of cell functions and interactions. TSPs in malignant tumors can both enhance and inhibit tumor progression, invasion, and metastasis, depending on cell type, stromal interactions, and microenvironment. These proteins are potential targets for anticancer therapy.

OBJECTIVE

The aim of our article is to review the role of thrombospondin-1 (TSP1) in cutaneous melanoma.

CONCLUSIONS

TSP1 expression is variable in melanoma cell lines and tumors. Similar to findings in other human cancers, expression of TSP1 by melanoma cells usually inhibits tumor progression via the antiangiogenic effect of TSP1. Conversely, stromal TSP1 overexpression in melanoma is a poor prognostic factor associated with decreased survival. Understanding the interactions of TSP1 with other melanoma- and matrix-associated proteins should provide new prognostic indices and possible therapeutic targets for melanoma treatment.

Acute lymphocytic leukemia (ALL) is the most common pediatric cancer. Currently, treatment options for patients with relapsed and refractory ALL mostly rely on immunotherapies. However, hematological cancers are commonly associated with a low immunogenicity and immune tolerance, which may contribute to leukemia relapse and the difficulties associated with the development of effective immunotherapies against this disease. We recently demonstrated that PKHB1, a TSP1-derived CD47 agonist peptide, induces immunogenic cell death (ICD) in T cell ALL (T-ALL). Cell death induced by PKHB1 on T-ALL cell lines and their homologous murine, L5178Y-R (T-murine tumor lymphoblast cell line), induced damage-associated molecular patterns (DAMPs) exposure and release. Additionally, a prophylactic vaccination with PKHB1-treated L5178Y-R cells prevented tumor establishment in vivo in all the cases. Due to the immunogenic potential of PKHB1-treated cells, in this study we assessed their ability to induce antitumor immune responses ex vivo and in vivo in an established tumor. We first confirmed the selectivity of cell death induced by PKBH1 in tumor L5178Y-R cells and observed that calreticulin exposure increased when cell death increased. Then, we found that the tumor cell lysate (TCL) obtained from PKHB1-treated L5178YR tumor cells (PKHB1-TCL) was able to induce, ex vivo, dendritic cells maturation, cytokine production, and T cell antitumor responses. Finally, our results show that in vivo, PKHB1-TCL treatment induces tumor regression in syngeneic mice transplanted with L5178Y-R cells, increasing their overall survival and protecting them from further tumor establishment after tumor rechallenge. Altogether our results highlight the immunogenicity of the cell death induced by PKHB1 activation of CD47 as a potential therapeutic tool to overcome the low immunogenicity and immune tolerance in T-ALL.

Human C8 is one of five components of the membrane attack complex of complement. It is an oligomeric protein composed of three subunits (C8 alpha, C8 beta, and C8 gamma) that are derived from different genes. C8 alpha and C8 beta are homologous and both contain a pair of tandemly arranged N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)], an extended middle segment referred to as the membrane attack complex/perforin region (MACPF), and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. During biosynthetic processing, C8 alpha and C8 gamma associate to form a disulfide-linked dimer (C8 alpha-gamma) that binds to C8 beta through a site located on C8 alpha. In this study, the location of binding sites for C8 beta and C8 gamma and the importance of the modules in these interactions were investigated by use of chimeric and truncated forms of C8 alpha in which module pairs were either exchanged for those in C8 beta or deleted. Results show that exchange or deletion of one or both pairs of modules does not abrogate the ability of C8 alpha to form a disulfide-linked dimer when coexpressed with C8 gamma in COS cells. Furthermore, each chimeric and truncated form of C8 alpha-gamma retains the ability to bind C8 beta; however, only those containing the TSP1 + LDLRA modules from C8 alpha are hemolytically active. These results indicate that binding sites for C8 beta and C8 gamma reside within the MACPF region of C8 alpha and that interaction with either subunit is not dependent on the modules. They also suggest that the N-terminal modules in C8 alpha are important for C9 binding and/or expression of C8 activity.

OBJECTIVE

Determine the safe dose of intravitreal propranolol (IVP), and evaluate its inhibitory effect on laser-induced choroidal neovascularization (CNV).

METHODS

To determine the IVP safe dose, 32 rabbits were divided into 4 groups. Three of these groups received IVP (15 μL) corresponding to 15 μg (group B), 30 μg (group C), and 60 μg (group D). The control group (A) received 15 μL saline. Safety was assessed by ocular examination, electroretinography (ERG), routine histopathologic evaluation, immunohistochemistry for glial fibrillary acidic protein (GFAP), and real-time qPCR for GFAP, VEGF, thrombospondin 1 (TSP1), and pigment epithelium-derived factor (PEDF). A similar experiment was performed in 24 mice by using a 100-fold lower amount of propranolol (0.15, 0.3, and 0.6 μg in 2 μL) based on vitreous volume. For assessment of the angioinhibitory effects of IVP, CNV was induced in 42 mice via laser burns. Mice were divided into two groups: group 1 received the safe dose of IVP (0.3 μg in 2 μL) and group 2 received saline. Neovascularization area was quantified by intercellular adhesion molecule (ICAM)-2 immunostaining of choroidal-scleral flat mounts by using ImageJ software.

RESULTS

According to clinical, ERG, and histopathologic findings, 30 μg IVP was chosen as the safe dose in rabbit eyes, comparable to 0.3 μg IVP in mouse eyes. As compared to the control eyes, the development of CNV was attenuated (4.8-fold) in mice receiving 0.3 μg IVP.

CONCLUSIONS

Intravitreal propranolol injection up to the final dose of 30 μg in rabbits and 0.3 μg in mice was safe, and was effective in attenuation of CNV in mice.

Abnormal stem cell function makes a known contribution to many malignant tumors, but the role of stem cells in benign tumors is not well understood. Here, we show that ossifying fibroma (OF) contains a stem cell population that resembles mesenchymal stem cells (OFMSCs) and is capable of generating OF-like tumor xenografts. Mechanistically, OFMSCs show enhanced TGF-β signaling that induces aberrant proliferation and deficient osteogenesis via Notch and BMP signaling pathways, respectively. The elevated TGF-β activity is tightly regulated by JHDM1D-mediated epigenetic regulation of thrombospondin-1 (TSP1), forming a JHDM1D/TSP1/TGF-β/SMAD3 autocrine loop. Inhibition of TGF-β signaling in OFMSCs can rescue their abnormal osteogenic differentiation and elevated proliferation rate. Furthermore, chronic activation of TGF-β can convert normal MSCs into OF-like MSCs via establishment of this JHDM1D/TSP1/TGF-β/SMAD3 autocrine loop. These results reveal that epigenetic regulation of TGF-β signaling in MSCs governs the benign tumor phenotype in OF and highlight TGF-β signaling as a candidate therapeutic target.

Macropinocytosis has been implicated in cardiovascular and other disorders, yet physiological factors that initiate fluid-phase internalization and the signaling mechanisms involved remain poorly identified. The present study was designed to examine whether matrix protein thrombospondin-1 (TSP1) stimulates macrophage macropinocytosis and, if so, to investigate the potential signaling mechanism involved.

TSP1 treatment of human and murine macrophages stimulated membrane ruffle formation and pericellular solute internalization by macropinocytosis. Blockade of TSP1 cognate receptor CD47 and NADPH oxidase 1 (Nox1) signaling, inhibition of phosphoinositide 3-kinase, and transcriptional knockdown of myotubularin-related protein 6 abolished TSP1-induced macropinocytosis. Our results demonstrate that Nox1 signaling leads to dephosphorylation of actin-binding protein cofilin at Ser-3, actin remodeling, and macropinocytotic uptake of unmodified native low-density lipoprotein (nLDL), leading to foam cell formation. Finally, peritoneal chimera studies suggest the role of CD47 in macrophage lipid macropinocytosis in hypercholesterolemic ApoE-/- mice in vivo.

Activation of a previously unidentified TSP1-CD47 signaling pathway in macrophages stimulates direct receptor-independent internalization of nLDL, leading to significant lipid accumulation and foam cell formation. These findings reveal a new paradigm in which delimited Nox1-mediated redox signaling, independent of classical lipid oxidation, contributes to early propagation of vascular inflammatory disease.

The findings of the present study demonstrate a new mechanism of solute uptake with implications for a wide array of cell types, including macrophages, dendritic cells, and cancer cells, and multiple pathological conditions in which matrix proteins are upregulated. Antioxid. Redox Signal. 26, 886-901.

Pseudoexfoliation (PEX) syndrome is a common, but little known, systemic degenerative condition manifest by the extracellular deposition of a distinctive fibrillar material (PEX material) in various organs. In the eye, PEX material is characteristically found on the surfaces of structures that line the anterior and posterior chambers, and it is associated with cataract and glaucoma. Although PEX material contains several elements normally present in basement membranes, its precise composition remains obscure. Because the glycoprotein thrombospondin 1 (TSP1) can be shown in some basement membranes, we attempted to define its involvement in the composition of PEX material by immunohistochemical analysis of ocular tissues from patients with PEX syndrome. Although we were unable to detect TSP1 in PEX material, we were surprised to find that iris and corneal stromal fibroblasts expressed TSP1. In age-matched normal eyes, iris and corneal fibroblasts did not contain demonstrable TSP1. These observations indicate that TSP1 is not a significant component of PEX material but suggest that, in PEX syndrome, stromal fibroblasts remote from the principal sites of PEX material deposition are altered at the molecular level. The findings add evidence to the theory that PEX syndrome represents a disorder of connective tissue metabolism and intimate that the syndrome involves anomalous production of proteins other than those found in PEX material.

OBJECTIVE

By the release of antiangiogenic factors, Müller glial cells provide an angiostatic environment in the normal and ischemic retina. We determined whether Müller cells produce thrombospondin-1 (TSP-1), a known inhibitor of angiogenesis.

METHODS

Secretion of TSP-1 by cultured Müller cells was determined with ELISA. Slices of rat retinas and surgically excised retinal membranes of human subjects were immunostained against TSP-1 and the glial marker vimentin. The effects of TSP-1 on the growth of bovine retinal endothelial cells (BRECs) and activation of ERK1/2 were determined with DNA synthesis and migration assays, and Western blotting, respectively.

RESULTS

Cultured Müller cells secrete TSP-1 under normoxic and hypoxic (0.2% O2) conditions. Secretion of TSP-1 was increased in hypoxia compared to normoxia. In rat retinal slices, glial, retinal ganglion, and possibly horizontal cells were stained for TSP-1. Retinal glial cells in preretinal membranes from human subjects with nonhypoxic epiretinal gliosis (macular pucker) and proliferative diabetic retinopathy, respectively, were immunopositive for TSP-1. Exogenous TSP-1 reduced the VEGF-induced proliferation and migration of BRECs and decreased the phosphorylation level of ERK1/2 in BRECs.

CONCLUSIONS

The data suggest that Müller cells are one major source of TSP-1 in the normal and ischemic retina. Glia-derived TSP1 may inhibit angiogenic responses in the ischemic retina.

VEGF signaling through VEGFR2 is a central regulator of the angiogenic response. Inhibition of VEGF signaling by the stress-induced matricellular protein TSP1 plays a role in modulating the angiogenic response to VEGF in both health and disease. TSP1 binding to CD47 inhibits VEGFR2 activation. The full implications of this inhibitory interaction are unknown. We developed a detailed rule-based computational model to inquire if TSP1-CD47 signaling through VEGF had downstream effects upon ERK1/2 and calcium. Our Simulations suggest that enhanced degradation of VEGFR2 initiated by the binding of TSP1 to CD47 is sufficient to explain the inhibition of VEGFR2 phosphorylation, calcium elevation, and ERK1/2 activation downstream of VEGF. A complementary mechanism involving the recruitment of phosphatases to the VEGFR2 complex with consequent increase in the rate of receptor dephosphorylation may augment the inhibition of the VEGF signal. The model was then utilized to simulate the effect of inhibiting external TSP1 or the depletion of CD47 as potential therapeutic strategies in restoring VEGF signaling. Results suggest that depleting CD47 is a more efficient strategy in inhibiting the effects of TSP1/CD47 on VEGF signaling. Our results highlight the utility of in silico investigations in elucidating and clarifying molecular mechanisms at the intersection of TSP1 and VEGF biology and in differentiating between competing pro-angiogenic therapeutic strategies relevant to peripheral arterial disease (PAD) and wound healing.

Antibody-mediated blockade of cluster of differentiation 47 (CD47)-thrombospondin-1 (TSP-1) interactions blocks osteoclast formation in vitro and attenuates parathyroid hormone (PTH)-induced hypercalcemia in vivo in mice. Hypercalcemia in this model reflects increased bone resorption. TSP-1 has two cell-associated binding partners, CD47 and CD36. The roles of these two molecules in mediating the effects of TSP1 in osteoclasts are unclear. Osteoclast formation was attenuated but not absent when preosteoclasts isolated from CD47-/- mice were cocultured with WT osteoblasts. Suppressing CD36 in osteoclast progenitors also attenuated osteoclast formation. The hypercalcemic response to a PTH infusion was blunted in CD47-/-/CD36-/- (double knockout (DKO)) female mice but not CD47-/- mice or CD36-/- animals, supporting a role for both CD47 and CD36 in mediating this effect. Consistent with this, DKO osteoclasts had impaired resorptive activity when analyzed in vitro Inhibition of nitric oxide (NO) signaling is known to promote osteoclastogenesis, and TSP-1 suppresses NO production and signaling. An anti-TSP-1 antibody increased NO production in osteoclasts, and the inhibitory effect of anti-TSP-1 on osteoclastogenesis was completely rescued by l-nitroarginine methyl ester (l-NAME), a competitive NO synthase inhibitor. Supportive of an important role for CD36 in mediating the pro-osteoclastogenic effects of TSP-1, engaging CD36 with a synthetic agonist, p907, suppressed NO production in anti-TSP-1-treated cultures, allowing osteoclast maturation to occur. These results establish that CD36 and CD47 both participate in mediating the actions of TSP-1 in osteoclasts and establish a physiologically relevant cross-talk in bone tissue between these two molecules.

A progressive decline in antioxidant potential and accumulation of reactive oxygen species (ROS) are major causes of pathogenesis of several diseases, including glaucoma. Trabecular meshwork (TM) dysfunction resulting in higher intraocular pressure (IOP) is a hallmark of glaucoma, but its causes are unclear. Using human (h) TM cells derived from glaucomatous and normal subjects of different ages and cells facing oxidative-stress, we showed that specific loss of moonlighting antioxidant protein Peroxiredoxin (Prdx) 6 in aging or in glaucomatous TM cells caused ROS accumulation and pathobiological changes in TM cells. Prdx6 limits the levels of ROS, thus preventing overstimulation of genes and resultant deleterious effects. We found that Prdx6 levels declined in aging and were reduced dramatically in glaucomatous and aged TM cells. Biochemical assays revealed enhanced levels of ROS, and high expression/activation of TGFβs and its responsive extracellular matrix genes α-SM, fibronectin, TGase2 and Tsp1 in aged or glaucomatous cells. Furthermore, hTM cells displayed typical features of the combined effects of TGFβs and oxidative-stress-induced cellular changes, showing increased levels of lipid peroxidation, oxidative DNA damage, and senescence markers p16, p21 and SA-βgal activity, along with reduced levels of telomerase expression and activity. Exposure to oxidative-stress (H2O2) or knocking down of Prdx6 (with antisense) accelerated this process. Importantly, Prdx6 delivery to sick or aged TM cells reversed the process. We propose Prdx6 as a potential therapeutic target to guard the TM from oxidative-stress and age-dependent accumulation of ROS by balancing redox-homeostasis to prevent ocular disorders, like glaucoma.

The multistep cleavage of carotenoids in Mucorales during the sexual phase results in a cocktail of trisporic acid (C18) sex pheromones. We hypothesized that the C18 trisporoid intermediates have a specific regulatory function for sex pheromone production and carotenogenesis that varies with genus/species and vegetative and sexual phases of their life cycles. Real-time quantitative PCR kinetics determined for Blakeslea trispora displayed a very high transcript turnover in the gene for carotenoid cleavage dioxygenase, tsp3, during the sexual phase. An in vivo enzyme assay and chromatographic analysis led to the identification of β-apo-12'-carotenal as the first apocarotenoid involved in trisporic acid biosynthesis in B. trispora. Supplementation of C18 trisporoids, namely D'orenone, methyl trisporate C, and trisporin C, increased tsp3 transcripts in the plus compared to minus partners. Interestingly, the tsp1 gene, which is involved in trisporic acid biosynthesis, was downregulated compared to tsp3 irrespective of asexual or sexual phase. Only the minus partners of both B. trispora and Mucor mucedo had enhanced β-carotene production after treatment with C20 apocarotenoids, 15 different trisporoids, and their analogues. We conclude that the apocarotenoids and trisporoids influence gene transcription and metabolite production, depending upon the fungal strain, corresponding genus, and developmental phase, representing a "chemical dialect" during sexual communication.

Thrombospondin-1 (TSP1), expressed in many cells and tissues is abundantly present in platelet alpha-granules, from where it is released upon platelet activation. Murine Tsp1(-/-) platelet studies have revealed that TSP1 is redundant for platelet aggregation, but that it reinforces platelet aggregate stabilization, especially in a shear field. von Willebrand factor (VWF), synthesized by megakaryocytes and endothelial cells is stored both in platelet alpha-granules and in endothelial Weibel-Palade bodies as ultralarge multimers. When released from endothelial cells, these multimers are temporarily retained on the endothelium, to be cleaved by the plasma protease ADAMTS13 into smaller and hemostatically less reactive multimers, released in plasma. This protease shows partial sequence identity with the type 1 (TSR1) and type 2 (TSR2) repeats of TSP1 and contains 1 TSR1 and 6 TSR2 repeats. TSP1, locally released by platelets, competes with ADAMTS13 during VWF proteolysis and controls the degree of VWF multimer processing. In addition, TSP1 and VWF both interact with the platelet GPIb/V/IX membrane complex, primarily in flow. These interactions control the recruitment of platelets to (sub) endothelial VWF and TSP1, exposed to the circulation, as a consequence of vascular inflammation and endothelial injury. TSP1-VWF interactions do not strictly enhance platelet recruitment and secreted TSP1 even weakly competes with the dynamic platelet rolling and adhesion onto VWF. Hence, TSP1 and VWF show partially related hemostatic functions, the most important one being the TSP1 role in the ADAMTS13 operated VWF multimer processing, in pro-inflammatory and thrombogenic conditions.

The extracellular matrix protein thrombospondin 1 (TSP1) was cloned from odontoblasts of bovine mandibular teeth which participate in dentinogenesis. The 5289 bp cDNA contains a complete open reading frame of 1170 amino acids. Bovine TSP1 has high homologies to its human and mouse counterparts. In immunohistochemical analyses of bovine anterior teeth with anti-TSP1 monoclonal antibody, TSP1 was only detectable at the position of predentin, located between dentin and unmineralized dental pulp. Northern blot analysis showed high levels of two sizes of TSP1 mRNAs in odontoblasts but not dental pulp and gingiva. Previously we found that osteotropic factors such as calcitriol and TGF-beta induce TSP1 at the transcriptional level in clonal rat dental pulp cells. These results suggest a role of TSP1 in dentinogenesis and/or maintenance of dentin and dental pulp.

Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin on the cell surface. Our previous studies indicate that uPA and uPAR expression increase in the ischemic brain during the recovery phase from an acute ischemic injury and that uPA binding to uPAR promotes neurological recovery after an acute ischemic stroke. Here, we used male mice genetically deficient on either uPA (uPA-/-) or uPAR (uPAR-/-) or with a four-amino acid substitution into the growth factor domain of uPA that abrogates its binding to uPAR (PlatGFDhu/GFDhu) to investigate the mechanism whereby uPA promotes neurorepair in the ischemic brain. We found that neurons release uPA and astrocytes recruit uPAR to their plasma membrane during the recovery phase from a hypoxic injury and that binding of neuronal uPA to astrocytic uPAR induces astrocytic activation by a mechanism that does not require plasmin generation, but instead is mediated by extracellular signal-regulated kinase 1/2 (ERK1/2)-regulated phosphorylation of the signal transducer and activator of transcription 3 (STAT3). We report that uPA/uPAR binding is necessary and sufficient to induce astrocytic activation in the ischemic brain and that astrocytes activated by neuronal uPA promote synaptic recovery in neurons that have suffered an acute hypoxic injury via a mechanism mediated by astrocytic thrombospondin-1 (TSP1) and synaptic low-density lipoprotein receptor-related protein-1 (LRP1). In summary, we show that uPA/uPAR-induced astrocytic activation mediates a cross talk between astrocytes and injured neurons that promotes synaptic recovery in the ischemic brain.SIGNIFICANCE STATEMENT To date, there is no therapeutic strategy to promote synaptic recovery in the injured brain. Here, we show that neurons release urokinase-type plasminogen activator (uPA) and astrocytes recruit the uPA receptor (uPAR) to their plasma membrane during the recovery phase from a hypoxic injury. We found that binding of neuronal uPA to astrocytic uPAR promotes astrocytic activation and that astrocytes activated by uPA-uPAR binding promote synaptic recovery in neurons that have suffered a hypoxic injury by a mechanism that does not require plasmin generation, but instead is mediated by ERK1/2-regulated STAT3 phosphorylation, astrocytic thrombospondin-1 (TSP1) and synaptic low-density lipoprotein receptor-related protein-1 (LRP1). Our work unveils a new biological function for uPA-uPAR as mediator of a neuron-astrocyte cross talk that promotes synaptic recovery in the ischemic brain.